Genetic Diversity and Multihost Pathogenicity of Clinical and Environmental Strains of Burkholderia cenocepacia

A collection of 54 clinical and agricultural isolates of Burkholderia cenocepacia was analyzed for genetic relatedness by using multilocus sequence typing (MLST), pathogenicity by using onion and nematode infection models, antifungal activity, and the distribution of three marker genes associated with virulence. The majority of clinical isolates were obtained from cystic fibrosis (CF) patients in Michigan, and the agricultural isolates were predominantly from Michigan onion fields. MLST analysis resolved 23 distinct sequence types (STs), 11 of which were novel. Twenty-six of 27 clinical isolates from Michigan were genotyped as ST-40, previously identified as the Midwest B. cenocepacia lineage. In contrast, the 12 agricultural isolates represented eight STs, including ST-122, that were identical to clinical isolates of the PHDC lineage. In general, pathogenicity to onions and the presence of the pehA endopolygalacturonase gene were detected only in one cluster of related strains consisting of agricultural isolates and the PHDC lineage. Surprisingly, these strains were highly pathogenic in the nematode Caenorhabditis elegans infection model, killing nematodes faster than the CF pathogen Pseudomonas aeruginosa PA14 on slow-kill medium. The other strains displayed a wide range of pathogenicity to C. elegans, notably the Midwest clonal lineage which displayed high, moderate, and low virulence. Most strains displayed moderate antifungal activity, although strains with high and low activities were also detected. We conclude that pathogenicity to multiple hosts may be a key factor contributing to the potential of B. cenocepacia to opportunistically infect humans both by increasing the prevalence of the organism in the environment, thereby increasing exposure to vulnerable hosts, and by the selection of virulence factors that function in multiple hosts.The betaproteobacterium Burkholderia cenocepacia, 1 of now 17 classified species belonging to the Burkholderia cepacia complex (BCC), is ubiquitous and extremely versatile in its metabolic capabilities and interactions with other organisms (38, 40, 57, 58). Strains of B. cenocepacia are pathogens of onion and banana plants, opportunistic pathogens of humans, symbionts of numerous plant rhizospheres, contaminants of pharmaceutical and industrial products, and inhabitants of soil and surface waters (14, 29, 33, 34, 37, 45). Originally described as a pathogen of onions (8), organisms of the BCC emerged in the past 3 decades as serious human pathogens, capable of causing devastating chronic lung infections in persons with cystic fibrosis (CF) or chronic granulomatous disease (21, 24, 28). Infections due to BCC are a serious concern to CF patients due to their inherent antibiotic resistance and high potential for patient-to-patient transmission (23). Although 16 of the BCC species have been recovered from respiratory secretions of CF patients in many countries (46, 58), B. cenocepacia has been the most common species isolated in North America, detected in 50% of 606, 83% of 447, and 45.6% of 1,218 patients in recent studies (35, 46, 52).The epidemiology of infectious disease caused by B. cenocepacia appears to involve patient-to-patient spread of genetically distinct lineages. B. cenocepacia lineages, such as ET12, Midwest, and PHDC, have been identified from large numbers of individuals in disease outbreaks in North America and Europe (11, 32, 54). A recently developed multilocus sequence typing (MLST) scheme has been shown to be a reliable epidemiologic tool for differentiating between the five subgroups (IIIA to IIIE) of B. cenocepacia, and strains representing three of these subgroups (IIIA, IIIB, and IIID) have been recovered from CF patients (2). Outside of the patient-to-patient transmission of clonal lineages, the mode of acquisition of strains causing sporadic cases of B. cenocepacia in CF patients remains unclear, although environmental sources are a logical reservoir for infection. Previously, an isolate of B. cenocepacia indistinguishable from the PHDC epidemic clonal lineage by using standard typing methods (e.g., repetitive-sequence-based PCR, randomly amplified polymorphic DNA, pulsed-field gel electrophoresis) was detected in an agricultural soil sample (34). Similarly, three distinct MLST sequence types containing both clinical and environmental (plant and soil) B. cenocepacia isolates were identified (1). These findings suggest that natural populations of B. cenocepacia in soil or associated with plants are a potential reservoir for the emergence of new human pathogenic lineages.Experimental models for the study of virulence potential and traits of B. cenocepacia include mouse and rat models with genetic defects allowing chronic lung infections to be established (e.g., see reference 48). Nematode (Caenorhabditis elegans), alfalfa (Medicago sativa), and onion (Allium cepa) models have also been routinely utilized for the identification of virulence factors (5, 29, 31). C. elegans has been extensively used to study the pathogenesis and virulence factors of a wide variety of bacterial and fungal pathogens (9, 15, 42, 51, 56). In several pathogens, including Pseudomonas (56) and Burkholderia (20), putative virulence factors important for the pathogenesis in mammalian systems (15, 51) have been identified using the C. elegans model. The C. elegans model might be limited in the detection of host-specific virulence factors; however, several attributes, such as small size and rapid development, make it an excellent whole animal model for pathogenesis research (16, 51).The evidence that individual strains of B. cenocepacia can be pathogenic to both plants and humans and are prevalent in various environmental niches has provoked particular interest in elucidating the clinical pathogenic potential of environmental isolates. The basis of this study was to examine whether genetically related B. cenocepacia strains exhibit shared characteristics that contribute to their pathogenicity in multiple hosts and to examine the potential for circulating environmental isolates to emerge as new clinical pathogens. Here, we tested the degree of virulence in animal (nematode) and plant (onion) infection models, the production of antifungal activity, and the genetic relatedness of clinical and environmental B. cenocepacia subgroup IIIB strains predominantly isolated from Michigan.     

Experimental Adaptation of Burkholderia cenocepacia to Onion Medium Reduces Host Range

It is unclear whether adaptation to a new host typically broadens or compromises host range, yet the answer bears on the fate of emergent pathogens and symbionts. We investigated this dynamic using a soil isolate of Burkholderia cenocepacia, a species that normally inhabits the rhizosphere, is related to the onion pathogen B. cepacia, and can infect the lungs of cystic fibrosis patients. We hypothesized that adaptation of B. cenocepacia to a novel host would compromise fitness and virulence in alternative hosts. We modeled adaptation to a specific host by experimentally evolving 12 populations of B. cenocepacia in liquid medium composed of macerated onion tissue for 1,000 generations. The mean fitness of all populations increased by 78% relative to the ancestor, but significant variation among lines was observed. Populations also varied in several phenotypes related to host association, including motility, biofilm formation, and quorum-sensing function. Together, these results suggest that each population adapted by fixing different sets of adaptive mutations. However, this adaptation was consistently accompanied by a loss of pathogenicity to the nematode Caenorhabditis elegans; by 500 generations most populations became unable to kill nematodes. In conclusion, we observed a narrowing of host range as a consequence of prolonged adaptation to an environment simulating a specific host, and we suggest that emergent pathogens may face similar consequences if they become host-restricted.Some emergent pathogens, such as Pseudomonas and Burkholderia species, persist in a wide range of plant and animal hosts, suggesting that the virulence factors needed to infect plants and animals are similar (5, 40). Yet whether adaptation to a new niche tends to compromise niche breadth or, in this case, host range is an open question. Adaptation to a novel host may restrict host range to various degrees, whether by diminishing host-specific virulence traits without affecting host colonization or by reducing the ability to initiate infection in alternative hosts. However, if factors needed to colonize plant and animal hosts are similar, then why are some bacterial populations restricted to a narrow host range while others are not? One explanation for a limited host range may be the result of genetic trade-offs associated with adaptation to a specific host (7, 18). Another explanation may be that prolonged adaptation to a specific host casts a “selective shadow” over unused functions that are relevant to colonizing other hosts but decay by genetic drift (7, 18). To address these possibilities, we quantified the direct and correlated effects of specific host adaptation by the opportunistic pathogen Burkholderia cenocepacia.Members of the Burkholderia cepacia complex (Bcc), which are ubiquitous in the environment, were once used as biocontrol and bioremedial agents but now are banned from these applications because of the potential of some members to cause plant and human disease (39). The type species B. cepacia is well known as a pathogen of the common yellow onion, Allium cepa, in which it causes a characteristic yellow or brown rot. Another species, B. cenocepacia, can also infect onions as well as a range of plants and animals, including humans (2, 6, 26, 36). Bcc bacteria can cause serious infection in the lungs of cystic fibrosis (CF) patients (6, 26). These infections, called “cepacia syndrome,” are highly contagious among CF patients, and infections produce many negative effects on an already poor quality of life, including longer hospital stays, removal from lung transplant lists, blood poisoning, and eventual death (24). B. cenocepacia, one of the two Bcc species most commonly isolated from lung infections, is especially threatening and is associated with more severe cepacia syndrome (35). However, the mechanisms allowing B. cenocepacia to adapt to colonize both human and plant hosts are unclear. Several putative virulence mechanisms have been identified by random mutagenic screens or by knockouts of candidate genes (2, 12, 20, 25, 29, 35, 43, 46), but these mechanisms generally have not been shown to function in host adaptation. One way to directly study adaptation of bacterial populations to susceptible hosts is by experimental evolution, in which bacterial populations evolve in a controlled laboratory setting that enables study of the adaptive process over time (7).We experimentally evolved populations of B. cenocepacia HI2424 to study the extent to which adaptation to the common yellow onion A. cepa affects host range. B. cenocepacia HI2424 is a soil isolate and is classified as part of the PHDC strain lineage, the strain first characterized as responsible for an outbreak of Bcc infections in large treatment centers located in the mid-Atlantic region of the United States (33). We found that adaptation of B. cenocepacia to the onion model was associated with reduced virulence but did not compromise the capacity to colonize (or be consumed by) the nematode Caenorhabditis elegans, and the coincidence of these events suggests that a genetic trade-off (antagonistic pleiotropy) between fitness in onion medium and nematode virulence exists. We also characterized several phenotypes potentially associated with adaptation to the onion or nematode virulence. Most phenotypes varied significantly among replicate populations, suggesting that adaptation to the onion model may follow several different pathways.     

Structural and Functional Characterization of Diffusible Signal Factor Family Quorum-Sensing Signals Produced by Members of the Burkholderia cepacia Complex

Previous work has shown that Burkholderia cenocepacia produces the diffusible signal factor (DSF) family signal cis-2-dodecenoic acid (C₁₂:Δ², also known as BDSF), which is involved in the regulation of virulence. In this study, we determined whether C₁₂:Δ² production is conserved in other members of the Burkholderia cepacia complex (Bcc) by using a combination of high-performance liquid chromatography, mass spectrometry, and bioassays. Our results show that five Bcc species are capable of producing C₁₂:Δ² as a sole DSF family signal, while four species produce not only C₁₂:Δ² but also a new DSF family signal, which was identified as cis,cis-11-methyldodeca-2,5-dienoic acid (11-Me-C₁₂:Δ^2,5). In addition, we demonstrate that the quorum-sensing signal cis-11-methyl-2-dodecenoic acid (11-Me-C₁₂:Δ²), which was originally identified in Xanthomonas campestris supernatants, is produced by Burkholderia multivorans. It is shown that, similar to 11-Me-C₁₂:Δ² and C₁₂:Δ², the newly identified molecule 11-Me-C₁₂:Δ^2,5 is a potent signal in the regulation of biofilm formation, the production of virulence factors, and the morphological transition of Candida albicans. These data provide evidence that DSF family molecules are highly conserved bacterial cell-cell communication signals that play key roles in the ecology of the organisms that produce them.The Burkholderia cepacia complex (Bcc) comprises a group of currently 17 formally named bacterial species that, although closely related, are phenotypically diverse (17, 22, 23). Strains of the Bcc are ubiquitously distributed in nature and have been isolated from soil, water, the rhizosphere of plants, industrial settings, hospital environments, and infected humans. Some Bcc strains have emerged as problematic opportunistic pathogens in patients with cystic fibrosis or chronic granulomatous disease, as well as in immunocompromised individuals (17). The clinical outcome of Bcc infections ranges from asymptomatic carriage to a fulminant and fatal pneumonia, the so-called “cepacia syndrome” (12, 17). Although all Bcc species have been isolated from both environmental and clinical sources, B. cenocepacia and B. multivorans are most commonly found in clinical samples (16).Many bacterial pathogens have evolved a cell-cell communication mechanism known as quorum sensing (QS) to coordinate the expression of virulence genes. In spite of their genetic differences, most Bcc species produce N-acylhomoserine lactone (AHL) QS signals (25). More recently, another QS signal molecule, cis-2-dodecenoic acid (BDSF), has been identified in B. cenocepacia (3). Subsequent studies showed that BDSF plays a role in the regulation of bacterial virulence (6, 20). Interestingly, the two QS systems appear to act in conjunction in the regulation of B. cenocepacia virulence, as a set of the AHL-controlled virulence genes are also positively regulated by BDSF (6). Furthermore, mutation of Bcam0581, which is required for BDSF biosynthesis, results in substantially retarded energy production and impaired growth in minimal medium (6), highlighting the dual roles of the QS system in the physiology of and infection by B. cenocepacia.BDSF is a structural analogue of cis-11-methyl-2-dodecenoic acid, which is a QS signal known as diffusible signal factor (DSF) originally identified from the plant bacterial pathogen Xanthomonas campestris pv. campestris (2, 24). Evidence is accumulating that DSF-type fatty acid signals represent a new family of QS signals, which are widespread among Gram-negative bacteria (10, 24). For example, DSF and seven structural derivatives were identified in supernatants of Stenotrophomonas maltophilia (8, 11), 12-methyl-tetradecanoic acid was shown to be produced by Xylella fastidiosa (18), and cis-2-decenocic acid was found to be synthesized by Pseudomonas aeruginosa (5). In addition, DSF-like activity has also been reported in a range of Xanthomonas species, including X. oryzae pv. oryzae and X. axonopodis pv. citri (1, 2, 4, 24), but the chemical structures of these DSF analogues remain to be determined. Unlike other known QS signals, such as AHL and AI-2 family signals, DSF and its analogues, including BDSF, are fatty acids and these fatty acid signals were collectively designated DSF family signals for the convenience of discussion (10). Considering the fact that the list of DSF family signal is expanding, we propose to designate cis-11-methyl-2-dodecenoic acid (DSF) 11-Me-C₁₂:Δ² and cis-2-dodecenoic acid (BDSF) C₁₂:Δ². This nomenclature is based on one of the fatty acid nomenclatures (13, 19) where the methyl (Me) substitution and its position are indicated first (for example, 11-Me indicates a methyl group on C-11 of the fatty acid carbon chain), followed by the length of the fatty acid carbon chain (C₁₂ represents a 12-carbon fatty acid chain), and then the position of the double bond in the fatty acid chain (Δ² indicates a double bond in the cis configuration at site 2, i.e., between C-2 and C-3 of the fatty acid carbon chain). In this way, it is convenient to say that 11-Me-C₁₂:Δ² and C₁₂:Δ² have identical 12-carbon fatty acid chains with a cis bond at the same site but differ in a methyl substitution on C-11. Following this nomenclature system, 12-methyl-tetradecanoic acid and cis-2-decenocic acid can be referred to as 12-Me-C₁₄ and C₁₀:Δ², respectively.DSF family signals have emerged as important factors in the regulation of virulence and biofilm formation in a wide range of bacterial pathogens (10). In this study, we have investigated the production of the DSF family signals in nine Bcc species. It is demonstrated that C₁₂:Δ² is conserved in members of the Bcc and that 11-Me-C₁₂:Δ² and a novel DSF family signal were also produced by some, but not all, of the Bcc strains investigated. This new signal was identified as cis,cis-11-methyldodeca-2,5-dienoic acid (11-Me-C₁₂:Δ^2,5) by nuclear magnetic resonance (NMR) analysis and mass spectrometry. We have also investigated the biological significance of 11-Me-C₁₂:Δ^2,5 in intraspecies and interspecies communication.     

Improved Electrotransformation and Decreased Antibiotic Resistance of the Cystic Fibrosis Pathogen Burkholderia cenocepacia Strain J2315

The bacterium Burkholderia cenocepacia is pathogenic for sufferers from cystic fibrosis (CF) and certain immunocompromised conditions. The B. cenocepacia strain most frequently isolated from CF patients, and which serves as the reference for CF epidemiology, is J2315. The J2315 genome is split into three chromosomes and one plasmid. The strain was sequenced several years ago, and its annotation has been released recently. This information should allow genetic experimentation with J2315, but two major impediments appear: the poor potential of J2315 to act as a recipient in transformation and conjugation and the high level of resistance it mounts to nearly all antibiotics. Here, we describe modifications to the standard electroporation procedure that allow routine transformation of J2315 by DNA. In addition, we show that deletion of an efflux pump gene and addition of spermine to the medium enhance the sensitivity of J2315 to certain commonly used antibiotics and so allow a wider range of antibiotic resistance genes to be used for selection.Burkholderia cenocepacia is part of the Burkholderia cepacia complex (Bcc), a group of closely related bacteria of soil, water, and roots (41) recently updated to at least 15 related species (42). Bcc displays many interesting features (see reference 27 for a review). Originally discovered as responsible for soft onion rot (3), Bcc species also interact beneficently with plants (see reference 34 for a review) and may degrade pollutants such as phthalate or the herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5,-T) (25, 33). But it is the emergence of Bcc as an opportunistic pathogen of people suffering from cystic fibrosis (CF) (19) and immunocompromizing conditions that has drawn most attention to these bacteria. Among Bcc species, Burkholderia multivorans and B. cenocepacia are the most prevalent in the epidemiology of CF. In particular, strains of the ET12 lineage of B. cenocepacia were responsible for a major transcontinental epidemic among CF patients in the 1990s (20), an outbreak aggravated by the high levels of resistance to nearly all antibiotics that characterizes Bcc. Species of the Bcc have large genomes (7 to 9 Mb) composed of two or three chromosomes and one or more plasmids, an unusual genomic organization among bacteria. The first Bcc genome to be sequenced was that of B. cenocepacia J2315 (also known as LMG16656), the type strain of the ET12 lineage and the reference strain for CF epidemiology; the sequence was completed and made available by the Wellcome Trust Sanger Institute in 2003. It revealed three chromosomes of 3.9, 3.2, and 0.9 Mb and a plasmid of 93 kb. The annotation of this genome was released recently (15).The pathogenicity and multipartite genome of B. cenocepacia make it an important subject for both practical and fundamental study. Genetic modification is essential to the success of many such investigations. Unfortunately, J2315 throws up major barriers to genetic manipulation. Standard electrotransformation techniques are ineffective with this strain, as also found elsewhere (26). Conjugal introduction of DNA has proved unreliable despite adaptations (7) that have enabled occasional successes with B. cenocepacia species (9, 40) including J2315 (39) (see also Results below). Besides, the natural resistance of J2315 to antibiotics, high even on the scale of the generally extensive resistance of B. cenocepacia species (31), severely restricts the use of antibiotic resistance in genetic selections. Circumventing these problems by resorting to a proxy strain, B. cenocepacia K56-2, that has not been sequenced and is more permissive to gene transfer (26, 17, 32, 9) runs the risk that results will be of uncertain relevance to J2315.In the context of our general aim to decipher the role of the four replicon-specific ParABS systems of J2315 (6), we have sought to overcome these obstacles. We report here the reproducible electrotransformation of J2315, and we analyze factors that improve its efficiency. We report also our isolation of a J2315 derivative with reduced antibiotic resistance and the broadened selection possibilities this offers. Detailed protocols are provided which should facilitate studies of this pathogen.     

Contribution of Lipoproteins and Lipoprotein Processing to Endocarditis Virulence in Streptococcus sanguinis

Streptococcus sanguinis is an important cause of infective endocarditis. Previous studies have identified lipoproteins as virulence determinants in other streptococcal species. Using a bioinformatic approach, we identified 52 putative lipoprotein genes in S. sanguinis strain SK36 as well as genes encoding the lipoprotein-processing enzymes prolipoprotein diacylglyceryl transferase (lgt) and signal peptidase II (lspA). We employed a directed signature-tagged mutagenesis approach to systematically disrupt these genes and screen each mutant for the loss of virulence in an animal model of endocarditis. All mutants were viable. In competitive index assays, mutation of a putative phosphate transporter reduced in vivo competitiveness by 14-fold but also reduced in vitro viability by more than 20-fold. Mutations in lgt, lspA, or an uncharacterized lipoprotein gene reduced competitiveness by two- to threefold in the animal model and in broth culture. Mutation of ssaB, encoding a putative metal transporter, produced a similar effect in culture but reduced in vivo competiveness by >1,000-fold. [³H]palmitate labeling and Western blot analysis confirmed that the lgt mutant failed to acylate lipoproteins, that the lspA mutant had a general defect in lipoprotein cleavage, and that SsaB was processed differently in both mutants. These results indicate that the loss of a single lipoprotein, SsaB, dramatically reduces endocarditis virulence, whereas the loss of most other lipoproteins or of normal lipoprotein processing has no more than a minor effect on virulence.Streptococcus sanguinis is a member of the viridans group of streptococci and is a primary colonizer of teeth (8). The viridans species and, in particular, S. sanguinis (15, 18) are a leading cause of infective endocarditis, a serious infection of the valves or lining of the heart (48). Damage to the heart resulting from rheumatic fever or certain congenital heart defects dramatically increases the risk of developing endocarditis (48, 71). The damage is thought to result in the formation of sterile cardiac “vegetations” composed of platelets and fibrin (48) that can be colonized by certain bacteria during periods of bacteremia. This view is supported by animal studies in which formation of sterile vegetation by cardiac catheterization is required for the efficient establishment of streptococcal endocarditis (17). Prevention of infective endocarditis currently relies upon prophylactic administration of antibiotics prior to dental or other surgical procedures that are likely to produce bacteremia. The growing realization that oral bacteria such as S. sanguinis can enter the bloodstream through routine daily activities such as eating has led the American Heart Association (71) and others (57) to question the value of using antibiotic prophylaxis for dental procedures. Clearly, a better understanding of the bacterial virulence factors that contribute to endocarditis could lead to better preventive measures, such as a vaccine that could potentially afford continuous protection to high-risk patients (71).In a previous study, we used the signature-tagged mutagenesis (STM) technique to search for endocarditis virulence factors of S. sanguinis in a rabbit model (53). This study identified a number of housekeeping enzymes that contribute to endocarditis. Because these proteins are not likely to be surface localized, they hold little promise as vaccine candidates. One class of streptococcal surface proteins that is rich in both virulence factors (4, 7, 25, 33, 38, 60) and promising vaccine candidates (6, 39, 42, 51, 70) is the lipoproteins. Lipoprotein activities that have been suggested to contribute to streptococcal virulence include adhesion (4, 7, 63), posttranslational modification (25, 29, 51), and ATP-binding cassette (ABC)-mediated transport (33, 52, 60). In the last instance, lipoproteins anchored to the cell membrane by their lipid tails appear to serve the same transport function as the periplasmic substrate-binding proteins of gram-negative bacteria (66). STM studies performed with Streptococcus pneumoniae (26, 41, 55) and Streptococcus agalactiae (34) have identified multiple lipoprotein mutants among collections of reduced virulence mutants. In an attempt to determine the cumulative contribution of streptococcal lipoproteins to virulence, some investigators have created mutations in the lgt or lspA genes, encoding lipoprotein-processing enzymes (12, 25, 27, 36). The lgt gene encodes prolipoprotein diacylglyceryl transferase, which catalyzes the transfer of a diacylglycerol lipid unit to a cysteine in the conserved N-terminal “lipobox” of lipoproteins, while lspA encodes the signal peptidase II enzyme that cleaves the signal peptide of the prolipoprotein just prior to the conserved cysteine (59, 65). While mutation of these genes has been shown to be lethal in gram-negative bacteria (21, 73), many gram-positive bacterial species have been shown to tolerate such mutations, often with only minor effects on growth (3, 12, 13, 25, 27, 36, 54). Some of these studies indicated a deleterious effect on the virulence of the lgt (25, 54) or lspA (36) mutation, but others found no effect (12) or an enhancement of virulence (27). It is clear from these and other studies (3, 13) that neither the loss of acylation due to lgt inactivation nor the loss of signal peptidase II-mediated cleavage completely eliminates lipoprotein function, necessitating alternative approaches for assessing the global contribution of lipoproteins to virulence.We have used bioinformatic approaches to identify every putative lipoprotein encoded by S. sanguinis strain SK36. To determine the contribution of these lipoproteins to the endocarditis virulence of S. sanguinis, we have systematically mutagenized each of these genes, as well as the lgt and lspA genes, and evaluated these mutants for virulence by using STM in an animal model. Selected mutants were further examined for virulence in competitive index (CI) assays. A strain with a disrupted ssaB gene, which encodes a putative metal transport protein, was found to exhibit a profound defect in virulence that was far greater than that of any other strain tested, including the lgt or lspA mutant.     

A New Borrelia Species Defined by Multilocus Sequence Analysis of Housekeeping Genes

Analysis of Lyme borreliosis (LB) spirochetes, using a novel multilocus sequence analysis scheme, revealed that OspA serotype 4 strains (a rodent-associated ecotype) of Borrelia garinii were sufficiently genetically distinct from bird-associated B. garinii strains to deserve species status. We suggest that OspA serotype 4 strains be raised to species status and named Borrelia bavariensis sp. nov. The rooted phylogenetic trees provide novel insights into the evolutionary history of LB spirochetes.Multilocus sequence typing (MLST) and multilocus sequence analysis (MLSA) have been shown to be powerful and pragmatic molecular methods for typing large numbers of microbial strains for population genetics studies, delineation of species, and assignment of strains to defined bacterial species (4, 13, 27, 40, 44). To date, MLST/MLSA schemes have been applied only to a few vector-borne microbial populations (1, 6, 30, 37, 40, 41, 47).Lyme borreliosis (LB) spirochetes comprise a diverse group of zoonotic bacteria which are transmitted among vertebrate hosts by ixodid (hard) ticks. The most common agents of human LB are Borrelia burgdorferi (sensu stricto), Borrelia afzelii, Borrelia garinii, Borrelia lusitaniae, and Borrelia spielmanii (7, 8, 12, 35). To date, 15 species have been named within the group of LB spirochetes (6, 31, 32, 37, 38, 41). While several of these LB species have been delineated using whole DNA-DNA hybridization (3, 20, 33), most ecological or epidemiological studies have been using single loci (5, 9-11, 29, 34, 36, 38, 42, 51, 53). Although some of these loci have been convenient for species assignment of strains or to address particular epidemiological questions, they may be unsuitable to resolve evolutionary relationships among LB species, because it is not possible to define any outgroup. For example, both the 5S-23S intergenic spacer (5S-23S IGS) and the gene encoding the outer surface protein A (ospA) are present only in LB spirochete genomes (36, 43). The advantage of using appropriate housekeeping genes of LB group spirochetes is that phylogenetic trees can be rooted with sequences of relapsing fever spirochetes. This renders the data amenable to detailed evolutionary studies of LB spirochetes.LB group spirochetes differ remarkably in their patterns and levels of host association, which are likely to affect their population structures (22, 24, 46, 48). Of the three main Eurasian Borrelia species, B. afzelii is adapted to rodents, whereas B. valaisiana and most strains of B. garinii are maintained by birds (12, 15, 16, 23, 26, 45). However, B. garinii OspA serotype 4 strains in Europe have been shown to be transmitted by rodents (17, 18) and, therefore, constitute a distinct ecotype within B. garinii. These strains have also been associated with high pathogenicity in humans, and their finer-scale geographical distribution seems highly focal (10, 34, 52, 53).In this study, we analyzed the intra- and interspecific phylogenetic relationships of B. burgdorferi, B. afzelii, B. garinii, B. valaisiana, B. lusitaniae, B. bissettii, and B. spielmanii by means of a novel MLSA scheme based on chromosomal housekeeping genes (30, 48).     

Absence of Escherichia coli Phylogenetic Group B2 Strains in Humans and Domesticated Animals from Jeonnam Province,Republic of Korea

Multiplex PCR analyses of DNAs from genotypically unique Escherichia coli strains isolated from the feces of 138 humans and 376 domesticated animals from Jeonnam Province, South Korea, performed using primers specific for the chuA and yjaA genes and an unknown DNA fragment, TSPE4.C2, indicated that none of the strains belonged to E. coli phylogenetic group B2. In contrast, phylogenetic group B2 strains were detected in about 17% (8 of 48) of isolates from feces of 24 wild geese and in 3% (3 of 96) of isolates obtained from the Yeongsan River in Jeonnam Province, South Korea. The distribution of E. coli strains in phylogenetic groups A, B1, and D varied depending on the host examined, and there was no apparent seasonal variation in the distribution of strains in phylogenetic groups among the Yeongsan River isolates. The distribution of four virulence genes (eaeA, hlyA, stx₁, and stx₂) in isolates was also examined by using multiplex PCR. Virulence genes were detected in about 5% (38 of 707) of the total group of unique strains examined, with 24, 13, 13, and 9 strains containing hlyA, eaeA, stx₂, and stx₁, respectively. The virulence genes were most frequently present in phylogenetic group B1 strains isolated from beef cattle. Taken together, results of these studies indicate that E. coli strains in phylogenetic group B2 were rarely found in humans and domesticated animals in Jeonnam Province, South Korea, and that the majority of strains containing virulence genes belonged to phylogenetic group B1 and were isolated from beef cattle. Results of this study also suggest that the relationship between the presence and types of virulence genes and phylogenetic groupings may differ among geographically distinct E. coli populations.Escherichia coli is a normal inhabitant of the lower intestinal tract of warm-blooded animals and humans. While the majority of E. coli strains are commensals, some are known to be pathogenic, causing intestinal and extraintestinal diseases, such as diarrhea and urinary tract infections (42). Phylogenetic studies done using multilocus enzyme electrophoresis and 72 E. coli strains in the E. coli reference collection showed that E. coli strains can be divided into four phylogenetic groups (A, B1, B2, and D) (20, 41, 48). Recently, a potential fifth group (E) has also been proposed (11). Since multiplex PCR was developed for analysis of phylogenetic groups (6), a number of studies have analyzed a variety of E. coli strains for their phylogenetic group association (10, 12, 17, 18, 23, 54). Duriez et al. (10) reported the possible influence of geographic conditions, dietary factors, use of antibiotics, and/or host genetic factors on the distribution of phylogenetic groups among 168 commensal E. coli strains isolated from human stools from three geographically distinct populations in France, Croatia, and Mali. Random-amplified polymorphic DNA analysis of the intraspecies distribution of E. coli in pregnant women and neonates indicated that there was a correlation between the distribution of phylogenetic groups, random-amplified polymorphic DNA groups, and virulence factors (54). Moreover, based on comparisons of the distribution of E. coli phylogenetic groups among humans of different sexes and ages, it has been suggested that E. coli genotypes are likely influenced by morphological, physiological, and dietary differences (18). In addition, climate has also been proposed to influence the distribution of strains within E. coli phylogenetic groups (12). There are now several reports indicating that there is a potential relationship between E. coli phylogenetic groups, age, and disease. For example, E. coli isolates belonging to phylogenetic group B2 have been shown to predominate in infants with neonatal bacterial meningitis (27) and among urinary tract and rectal isolates (55). Also, Nowrouzian et al. (39) and Moreno et al. (37) reported that strains belonging to phylogenetic group B2 persisted among the intestinal microflora of infants and were more likely to cause clinical symptoms.Boyd and Hartl (2) reported that among the E. coli strains in the E. coli reference and the diarrheagenic E. coli collections, strains in phylogenetic group B2 carry the greatest number of virulence factors, followed by those in group D. Virulence factors carried by group B2 strains are thought to contribute to their strong colonizing capacity; a greater number of virulence genes have been detected in resident strains than in transient ones (38). Moreover, a mouse model of extraintestinal virulence showed that phylogenetic group B2 strains killed mice at greater frequency and possessed more virulence determinants than strains in other phylogenetic groups, suggesting a link between phylogeny and virulence genes in E. coli extraintestinal infection (45). In contrast, Johnson and Kuskowski (25) suggested that a group B2 ancestral strain might have simply acquired virulence genes by chance and that these genes were vertically inherited by group members during clonal expansion. However, numerous studies published to date suggest that there is a relationship between the genomic background of phylogenetic group B2 and its association with virulence factors (12, 28, 35, 39, 45).Both enteropathogenic and enterohemorrhagic E. coli (EPEC and EHEC, respectively) strains are among the most important food-borne pathogens worldwide, often causing severe gastrointestinal disease and fatal infections (13). While EPEC strains cause diarrhea and generally do not produce enterotoxin, they possess an adherence factor which is controlled by the chromosomal gene eaeA, encoding intimin (8). Unlike the EPEC strains, however, the EHEC strains typically contain the hlyA, stx₁, and stx₂ virulence genes, encoding hemolysins and Shiga-like type 1 and 2 toxins, respectively, and eaeA. The ability to detect EHEC has been greatly facilitated by the use of multiplex PCR (13, 44, 53). Several studies have shown that strains producing Shiga-like toxin 2 are more frequently found in cases of hemolytic-uremic syndrome than are those containing Shiga-like toxin 1 (30, 43, 46, 49).In the study reported here, we examined the distribution of phylogenetic groups and the prevalence of virulence genes in 659 genotypically unique E. coli strains isolated from humans and domestic animals in South Korea. In addition, we also tested 48 and 96 nonunique E. coli isolates from wild geese and the Yeongsan River, respectively, for phylogenetic distribution and virulence gene profiles. Here, we report that contrary to what has been previously reported in other parts of the world, no E. coli strains belonging to phylogenetic group B2 were found in domesticated animals and in humans from Jeonnam Province, South Korea. We also report that among the strains we examined, virulence genes were mainly found in phylogenetic group B1 strains isolated from beef cattle. Results of these studies may prove to be useful for the development of risk management strategies to maintain public health.     

Thioredoxin 1 Participates in the Activity of the Salmonella enterica Serovar Typhimurium Pathogenicity Island 2 Type III Secretion System

The facultative intracellular pathogen Salmonella enterica serovar Typhimurium relies on its Salmonella pathogenicity island 2 (SPI2) type III secretion system (T3SS) for intracellular replication and virulence. We report that the oxidoreductase thioredoxin 1 (TrxA) and SPI2 are coinduced for expression under in vitro conditions that mimic an intravacuolar environment, that TrxA is needed for proper SPI2 activity under these conditions, and that TrxA is indispensable for SPI2 activity in both phagocytic and epithelial cells. Infection experiments in mice demonstrated that SPI2 strongly contributed to virulence in a TrxA-proficient background whereas SPI2 did not affect virulence in a trxA mutant. Complementation analyses using wild-type trxA or a genetically engineered trxA coding for noncatalytic TrxA showed that the catalytic activity of TrxA is essential for SPI2 activity in phagocytic cells whereas a noncatalytic variant of TrxA partially sustained SPI2 activity in epithelial cells and virulence in mice. These results show that TrxA is needed for the intracellular induction of SPI2 and provide new insights into the functional integration between catalytic and noncatalytic activities of TrxA and a bacterial T3SS in different settings of intracellular infections.In Escherichia coli, thioredoxin 1 (TrxA, encoded by trxA) is an evolutionary conserved 11-kDa cytosolic highly potent reductase that supports the activities of various oxidoreductases and ribonucleotide reductases (1, 29) and interacts with a number of additional cytoplasmic proteins through the formation of temporary covalent intermolecular disulphide bonds (32). Consequently, as trxA mutants of E. coli (51), Helicobacter pylori (13), and Rhodobacter sphaeroides (34) show increased sensitivity to hydrogen peroxide, TrxA has been defined as a significant oxidoprotectant. In addition, TrxA possess a protein chaperone function that is disconnected from cysteine interactions (30, 32).Salmonella enterica serovar Typhimurium is closely related to E. coli. During divergent evolution, the Salmonella genome acquired a number of virulence-associated genes (20). Many of these genes are clustered on genetic regions termed Salmonella pathogenicity islands (or SPIs). Of these, SPI1 and SPI2 code for separate type III secretion systems (T3SSs). T3SSs are supramolecular virulence-associated machineries that, in several pathogenic gram-negative bacterial species, enable injection of effector proteins from the bacteria into host cells (22, 57). The effector proteins, in turn, manipulate intrinsic host cell functions to facilitate the infection.The SPI1 T3SS of S. serovar Typhimurium is activated for expression in the intestine in response to increased osmolarity and decreased oxygen tension (22, 57). SPI1 effector proteins are primarily secreted into cells that constitute the epithelial layer and interfere with host cell Cdc42 and Rac-1 signaling and actin polymerization. This enables the bacteria to orchestrate their own actin-dependent uptake into nonphagocytic cells (57). SPI1 effector proteins also induce inflammatory signaling and release of interleukin-1β from infected cells (25, 26).Subsequent systemic progression of S. serovar Typhimurium from the intestinal tissue relies heavily on an ability to survive and replicate in phagocytic cells (18, 46, 53, 54). S. serovar Typhimurium uses an additional set of effector proteins secreted by the SPI2 T3SS for replication inside host cells and for coping with phagocyte innate responses to the infection (10, 11, 54). The functions of SPI2 effectors include diversion of vesicular trafficking, induction of apoptotic responses, and manipulation of ubiquitination of host proteins (28, 40, 45, 53). Hence, SPI2 effector proteins create a vacuolar environment that sustains intracellular replication of S. serovar Typhimurium (28).In addition to pathogenicity islands, the in vivo fitness of Salmonella spp. relies on selected functions shared with other enterobacteria. Thus, many virulence genes are integrated into “housekeeping” gene regulatory networks, coded for by a core genome, which steer bacterial stress responses (12, 17, 27, 55). Selected anabolic pathways also contribute to virulence of S. serovar Typhimurium (18, 27), evidently by providing biochemical building blocks for bacterial replication (36).In S. serovar Typhimurium, TrxA is a housekeeping protein that strongly contributes to virulence in cell culture and mouse infection models (8). However, the mechanism by which TrxA activity adds to virulence has not been defined. Here we show that the contribution of TrxA to virulence of S. serovar Typhimurium associates with its functional integration with the SPI2 T3SS under conditions that prevail in the intracellular vacuolar compartment of the host cell. These findings ascribe a novel role to TrxA in bridging environmental adaptations with virulence gene expression and illuminate a new aspect of the interaction between evolutionary conserved and horizontally acquired gene functions in bacteria.     

Population Structure of the Lyme Borreliosis Spirochete Borrelia burgdorferi in the Western Black-Legged Tick (Ixodes pacificus) in Northern California

Factors potentially contributing to the lower incidence of Lyme borreliosis (LB) in the far-western than in the northeastern United States include tick host-seeking behavior resulting in fewer human tick encounters, lower densities of Borrelia burgdorferi-infected vector ticks in peridomestic environments, and genetic variation among B. burgdorferi spirochetes to which humans are exposed. We determined the population structure of B. burgdorferi in over 200 infected nymphs of the primary bridging vector to humans, Ixodes pacificus, collected in Mendocino County, CA. This was accomplished by sequence typing the spirochete lipoprotein ospC and the 16S-23S rRNA intergenic spacer (IGS). Thirteen ospC alleles belonging to 12 genotypes were found in California, and the two most abundant, ospC genotypes H3 and E3, have not been detected in ticks in the Northeast. The most prevalent ospC and IGS biallelic profile in the population, found in about 22% of ticks, was a new B. burgdorferi strain defined by ospC genotype H3. Eight of the most common ospC genotypes in the northeastern United States, including genotypes I and K that are associated with disseminated human infections, were absent in Mendocino County nymphs. ospC H3 was associated with hardwood-dominated habitats where western gray squirrels, the reservoir host, are commonly infected with LB spirochetes. The differences in B. burgdorferi population structure in California ticks compared to the Northeast emphasize the need for a greater understanding of the genetic diversity of spirochetes infecting California LB patients.In the United States, Lyme borreliosis (LB) is the most commonly reported vector-borne illness and is caused by infection with the spirochete Borrelia burgdorferi (3, 9, 52). The signs and symptoms of LB can include a rash, erythema migrans, fever, fatigue, arthritis, carditis, and neurological manifestations (50, 51). The black-legged tick, Ixodes scapularis, and the western black-legged tick, Ixodes pacificus, are the primary vectors of B. burgdorferi to humans in the United States, with the former in the northeastern and north-central parts of the country and the latter in the Far West (9, 10). These ticks perpetuate enzootic transmission cycles together with a vertebrate reservoir host such as the white-footed mouse, Peromyscus leucopus, in the Northeast and Midwest (24, 35), or the western gray squirrel, Sciurus griseus, in California (31, 46).B. burgdorferi is a spirochete species with a largely clonal population structure (14, 16) comprising several different strains or lineages (8). The polymorphic ospC gene of B. burgdorferi encodes a surface lipoprotein that increases expression within the tick during blood feeding (47) and is required for initial infection of mammalian hosts (25, 55). To date, approximately 20 North American ospC genotypes have been described (40, 45, 49, 56). At least four, and possibly up to nine, of these genotypes are associated with B. burgdorferi invasiveness in humans (1, 15, 17, 49, 57). Restriction fragment length polymorphism (RFLP) and, subsequently, sequence analysis of the 16S-23S rRNA intergenic spacer (IGS) are used as molecular typing tools to investigate genotypic variation in B. burgdorferi (2, 36, 38, 44, 44, 57). The locus maintains a high level of variation between related species, and this variation reflects the heterogeneity found at the genomic level of the organism (37). The IGS and ospC loci appear to be linked (2, 8, 26, 45, 57), but the studies to date have not been representative of the full range of diversity of B. burgdorferi in North America.Previous studies in the northeastern and midwestern United States have utilized IGS and ospC genotyping to elucidate B. burgdorferi evolution, host strain specificity, vector-reservoir associations, and disease risk to humans. In California, only six ospC and five IGS genotypes have been described heretofore in samples from LB patients or I. pacificus ticks (40, 49, 56) compared to approximately 20 ospC and IGS genotypes identified in ticks, vertebrate hosts, or humans from the Northeast and Midwest (8, 40, 45, 49, 56). Here, we employ sequence analysis of both the ospC gene and IGS region to describe the population structure of B. burgdorferi in more than 200 infected I. pacificus nymphs from Mendocino County, CA, where the incidence of LB is among the highest in the state (11). Further, we compare the Mendocino County spirochete population to populations found in the Northeast.     

Carboxylate Transporter Gene JEN1 from the Entomopathogenic Fungus Beauveria bassiana Is Involved in Conidiation and Virulence

Beauveria bassiana is an important entomopathogenic fungus widely used as a biological agent to control insect pests. A gene (B. bassiana JEN1 [BbJEN1]) homologous to JEN1 encoding a carboxylate transporter in Saccharomyces cerevisiae was identified in a B. bassiana transfer DNA (T-DNA) insertional mutant. Disruption of the gene decreased the carboxylate contents in hyphae, while increasing the conidial yield. However, overexpression of this transporter resulted in significant increases in carboxylates and decreased the conidial yield. BbJEN1 was strongly induced by insect cuticles and highly expressed in the hyphae penetrating insect cuticles not in hyphal bodies, suggesting that this gene is involved in the early stage of pathogenesis of B. bassiana. The bioassay results indicated that disruption of BbJEN1 significantly reduced the virulence of B. bassiana to aphids. Compared to the wild type, ΔBbJEN1 alkalinized the insect cuticle to a reduced extent. The alkalinization of the cuticle is a physiological signal triggering the production of pathogenicity. Therefore, we identified a new factor influencing virulence, which is responsible for the alkalinization of the insect cuticle and the initiation of fungal pathogenesis in insects.Mycoinsecticides are considered promising biological control agents and alternatives or supplements to chemical pesticides (15). However, the dearth of physiological, genetic, and molecular knowledge of entomopathogenic fungi has retarded their widespread application.For mycoinsecticide improvement, greater attention and effort have been given to elucidate the mechanisms of fungal pathogenesis (13, 14, 18, 20, 29, 49, 50, 51, 52, 53). Entomopathogenic fungi, e.g., Metarhizium anisopliae and Beauveria bassiana, invade their hosts by direct penetration of the host exoskeleton or cuticle. M. anisopliae and B. bassiana produce hydrophobic spores which contact and adhere to the insect cuticle (12). Once attached, the conidium germinates and the germ tubes differentiate into swollen infection structures called appressoria. The appressoria produce penetration pegs which penetrate the insect cuticle via cuticle-degrading enzymes (11, 19, 46) as well as mechanical pressure (24, 53). Hyphae proliferate within the hemocoel, emerge from inside the insect, and subsequently conidiate on the cadaver (15). However, much remains to be elucidated regarding the mechanisms of insect fungal pathogenesis.To obtain detailed knowledge of the mechanisms of fungal pathogenesis, a pool of B. bassiana transfer DNA (T-DNA) insertional mutants had been generated through an Agrobacterium-mediated-transformation method (21). A mutant, designated T12, characterized by the presence of more conidia, was isolated, and its flanking sequence was obtained by T-DNA tagging. The flanking fragment contained an open reading frame (ORF), which corresponded to a gene termed JEN1, encoding a transporter of carboxylates (http://www.ncbi.nlm.nih.gov/Blast.cgi). Organic acid transportation is important for the metabolism of almost all cells of multicellular organisms and unicellular microorganisms (17, 25, 26). Transport across the plasma membrane is the first step in the metabolism of these substrates, which may affect many aspects of the organism, including regulation of energy metabolism (9, 34) and acid-base equilibrium status (10).JEN1p has been identified in several fungal species, e.g., Saccharomyces cerevisiae, Candida albicans, and Kluyveromyces lactis (9, 35, 45), which is a lactate/pyruvate symporter (1, 9, 34). The enzyme imports lactate or some short-chain monocarboxylates across the plasma membrane into cells. Then, the lactate is stereo-specifically oxidized to pyruvate. This reaction is performed by ferricytochrome c oxidoreductase in mitochondria (23, 33) and is tightly connected to the respiratory chain (34). JEN1 was induced by lactic, pyruvic, acetic, and propionic acids and repressed by glucose (2, 9, 35, 45). Nevertheless, for entomopathogenic fungi, the characterization of JEN1p has not been investigated, and its role in infection is still a mystery.For this paper, we studied the functions of a putative carboxylate transport gene, JEN1, in B. bassiana (BbJEN1). Our results demonstrated that BbJEN1 is involved in conidiation of B. bassiana and that the gene is a new factor influencing virulence in entomopathogenic fungi.     

Roles of the Glyoxylate and Methylcitrate Cycles in Sexual Development and Virulence in the Cereal Pathogen Gibberella zeae

The glyoxylate and methylcitrate cycles are involved in the metabolism of two- or three-carbon compounds in fungi. To elucidate the role(s) of these pathways in Gibberella zeae, which causes head blight in cereal crops, we focused on the functions of G. zeae orthologs (GzICL1 and GzMCL1) of the genes that encode isocitrate lyase (ICL) and methylisocitrate lyase (MCL), respectively, key enzymes in each cycle. The deletion of GzICL1 (ΔGzICL1) caused defects in growth on acetate and in perithecium (sexual fruiting body) formation but not in virulence on barley and wheat, indicating that GzICL1 acts as the ICL of the glyoxylate cycle and is essential for self-fertility in G. zeae. In contrast, the ΔGzMCL1 strains failed to grow on propionate but exhibited no major changes in other traits, suggesting that GzMCL1 is required for the methylcitrate cycle in G. zeae. Interestingly, double deletion of both GzICL1 and GzMCL1 caused significantly reduced virulence on host plants, indicating that both GzICL1 and GzMCL1 have redundant functions for plant infection in G. zeae. Thus, both GzICL1 and GzMCL1 may play important roles in determining major mycological and pathological traits of G. zeae by participating in different metabolic pathways for the use of fatty acids.During the infection process, pathogenic fungi usually encounter nutrient deprivation in the host before gaining access to sufficient nutrients for successful colonization of the living tissue. To cope with a nutrient-limited environment, fungal pathogens seem to rely mostly on fatty acid metabolism for both energy supply and biosynthesis of essential molecules (29). The ability of fungi to use fatty acids as a carbon source for growth is based on the glyoxylate cycle. Fungal pathogens have been proposed to employ the glyoxylate bypass for the use of acetyl coenzyme A (CoA) units produced by the β-oxidation of even-chain-length fatty acids, probably available from host cell membranes or the lipid reservoir inside the fungal spore (7, 12, 20, 27, 28, 41, 44, 46). Recent studies suggest that the glyoxylate pathway plays an important role in fungal virulence toward both plant and animal hosts (12, 20, 27, 44, 46). The key enzymes of the glyoxylate pathway, such as isocitrate lyase (ICL), which catalyzes the cleavage of isocitrate to glyoxylate and succinate, and malate synthase, which mediates the condensation of acetyl-CoA and glyoxylate into malate, are strongly induced within the host (16, 27, 41, 44). Moreover, disruption of genes encoding either of these enzymes causes severely reduced virulence of fungal phytopathogens, including Leptosphaeria maculans (20), Magnaporthe grisea (46), Stagonospora nodorum (44), and Colletotrichum lagenarium (2), and the animal pathogen Candida albicans (27). In contrast, these glyoxylate cycle enzymes have been known to be dispensable in invasive aspergillosis caused by Aspergillus fumigatus (38, 43).During fatty acid and amino acid catabolism by fungi, propionyl-CoA can be generated along with acetyl-CoA, particularly from the breakdown of odd-chain-length fatty acids or of the amino acids valine, isoleucine, and methionine (14). Therefore, fungal pathogens may need to use or remove propionyl-CoA during the infection process because it is toxic to fungi. In fungi, propionyl-CoA is metabolized via the methylcitrate cycle, in which propionyl-CoA is oxidized to pyruvate in four enzymatic steps (4, 5, 6, 19, 30, 31, 40, 49, 50). Recently, the importance of the methylcitrate cycle in fungal virulence was demonstrated in A. fumigatus: a mutant defective in methylcitrate synthase, the first enzyme of this cycle, displayed attenuated virulence in mice and insects (19, 31). However, the role of methylisocitrate lyase (MCL), which catalyzes the last reaction in the methylcitrate cycle (i.e., the cleavage of methylisocitrate into pyruvate and succinate) in fungal virulence, has not been determined, although deletion of the MCL gene inhibits hyphal growth and conidiation in Aspergillus nidulans (4). The protein sequences of several fungal MCLs show high similarity to fungal ICLs of the glyoxylate cycle (4, 30). In the pathogenic bacterium Mycobacterium tuberculosis, the methylcitrate cycle, only when working together with the glyoxylate cycle, is involved in virulence as well as fatty acid metabolism and intracellular growth (34, 35).Here, we focused on the roles of these two cycles during disease development caused by the devastating cereal pathogen Gibberella zeae (anamorph: Fusarium graminearum). G. zeae is a ubiquitously distributed ascomycete fungus that causes major disease in cereal crops such as corn, wheat, barley, and rice (33). Severe epidemics of these diseases result in serious economic consequences due to yield losses and contamination by fungal mycotoxins (32, 33). Wind-disseminated sexual spores (ascospores), which are produced in perithecia formed on plant debris, can infect plant spikes during anthesis (13, 39, 45). Detailed studies of the G. zeae infection process on wheat and barley heads have shown that fungal hyphae on the inner surfaces of the spike penetrate epicarp cells through pits or pores and grow into the caryopses through the pericarp (21). Thus, the glyoxylate cycle, either alone or in conjunction with the methylcitrate cycle, is likely employed by G. zeae during the infection process, as in other fungus-plant interactions (20, 46). G. zeae genome searches have identified orthologs of fungal ICL and MCL genes, designated GzICL1 and GzMCL1, respectively. Here, we performed functional analyses of these genes to provide new insight into their importance in lipid metabolism during the G. zeae infection process in host plants.     

Inactivation of Burkholderia cepacia Complex Phage KS9 gp41 Identifies the Phage Repressor and Generates Lytic Virions

The Burkholderia cepacia complex (BCC) is made up of at least 17 species of Gram-negative opportunistic bacterial pathogens that cause fatal infections in patients with cystic fibrosis and chronic granulomatous disease. KS9 (vB_BcenS_KS9), one of a number of temperate phages isolated from BCC species, is a prophage of Burkholderia pyrrocinia LMG 21824. Transmission electron micrographs indicate that KS9 belongs to the family Siphoviridae and exhibits the B1 morphotype. The 39,896-bp KS9 genome, comprised of 50 predicted genes, integrates into the 3′ end of the LMG 21824 GTP cyclohydrolase II open reading frame. The KS9 genome is most similar to uncharacterized prophage elements in the genome of B. cenocepacia PC184 (vB_BcenZ_ PC184), as well as Burkholderia thailandensis phage φE125 and Burkholderia pseudomallei phage φ1026b. Using molecular techniques, we have disrupted KS9 gene 41, which exhibits similarity to genes encoding phage repressors, producing a lytic mutant named KS9c. This phage is incapable of stable lysogeny in either LMG 21824 or B. cenocepacia strain K56-2 and rescues a Galleria mellonella infection model from experimental B. cenocepacia K56-2 infections at relatively low multiplicities of infection. These results readily demonstrate that temperate phages can be genetically engineered to lytic form and that these modified phages can be used to treat bacterial infections in vivo.The Burkholderia cepacia complex (BCC) is a group of at least 17 Gram-negative species, the first identified strains of which were characterized as onion pathogens by W. H. Burkholder (9). Although these bacteria have a number of beneficial activities, including the promotion of crop growth and the degradation of organic pollutants, they have gained notoriety in the last two decades as serious opportunistic pathogens (19, 21, 25). BCC species, particularly B. multivorans and B. cenocepacia, cause serious respiratory infections in patients with cystic fibrosis and chronic granulomatous disease (42, 7). These infections are especially problematic due to symptom severity, the inherent antibiotic resistance of Bcc species, and the potential for rapid spread through susceptible patient populations (25, 23). Difficulties in treating these infections have led to the unfortunate practice of segregating patients, which has high economic, social, and psychological costs (18).Because of these clinical difficulties, interest in the isolation and characterization of Burkholderia-specific bacteriophages (or phages) has increased in recent years, with the apparent potential for using phages as therapeutic agents. Phage therapy is the clinical application of phages to prevent and/or to treat infections, which offers a promising alternative to antibiotic treatment for resistant bacteria such as those of the BCC (33, 39). A second benefit of these phage studies is that they may provide insight into the possible mechanisms of BCC virulence. For example, BcepMu, a transposable phage that specifically infects strains of B. cenocepacia, was found to carry genes similar to exeA, involved in toxin secretion, and mdmB and oafA, two acyltransferases (44). Finally, as Burkholderia phages tend to be underrepresented in comparative studies with respect to Escherichia coli and lactic acid bacteria phages, BCC-specific phage studies provide novel information about a relatively uncharacterized group of viruses.Although phage therapy using temperate virions can be effective (39), there are several reasons why lytic phages are generally considered the most appropriate candidates for use in phage therapy. One of the concerns is that phage integration can lead to lysogenic conversion and enhanced virulence (8). A second concern is that integration of temperate phages results in superinfection immunity due to expression of the phage repressor from the prophage. This protein binds to the operators of infecting phage DNA and represses gene expression, preventing both the initiation of the lytic cycle and the establishment of lysogeny (14). A third concern is that lysogeny affects the kinetics of infection. When a phage infects a cell and undergoes lysogeny instead of entering the lytic cycle, the cell survives, and no new phage particles are released (27). A final problem is that prophages can lead to specialized transduction after induction. Specialized transduction occurs after inexact excision of a prophage from the bacterial chromosome. Bacterial DNA flanking the prophage is packaged into the capsid, and this sequence, which can potentially encode virulence factors, can subsequently recombine into the chromosome of a new host (14).It has been estimated that more than half of tailed phages have evolved a temperate lifestyle, although some estimates have been greater than 90% (1, 22). This situation makes the isolation of naturally lytic phages extremely difficult, particularly when they must have a specific host range that includes clinically relevant bacterial species, such as B. cenocepacia (24). The use of classical genetics to produce lytic phage variants, for example, by plating temperate phages on lysogens and screening for clear plaque vir mutants, is complicated by the fact that such mutations are undefined.This report describes the characterization of KS9 (vB_BcenS_KS9), a prophage of Burkholderia pyrrocinia LMG 21824 (41), and its conversion to a lytic phage through specific molecular modification of gene 41 encoding its putative lytic phase repressor. Preliminary characterization of short sequences by Seed and Dennis (41) indicated that the genome of KS9, whose host range includes Bcc B. cenocepacia K56-2, shows similarity to the genomes of two non-BCC Burkholderia phages: φE125, a prophage of Burkholderia thailandensis E125 (47), and φ1026b, a prophage of Burkholderia pseudomallei 1026b (17). However, no phages closely related to KS9 have been functionally tested to demonstrate that proteins similar to gp41 function as true phage repressors. In the present study, we have used the BCC infection model of Galleria mellonella (40) to assess both the contribution of the KS9 prophage to BCC host virulence and the ability of a genetically modified KS9 to treat B. cenocepacia infections without stably integrating into the host bacterial chromosome as a prophage.     

A Bacillus anthracis S-Layer Homology Protein That Binds Heme and Mediates Heme Delivery to IsdC

The sequestration of iron by mammalian hosts represents a significant obstacle to the establishment of a bacterial infection. In response, pathogenic bacteria have evolved mechanisms to acquire iron from host heme. Bacillus anthracis, the causative agent of anthrax, utilizes secreted hemophores to scavenge heme from host hemoglobin, thereby facilitating iron acquisition from extracellular heme pools and delivery to iron-regulated surface determinant (Isd) proteins covalently attached to the cell wall. However, several Gram-positive pathogens, including B. anthracis, contain genes that encode near iron transporter (NEAT) proteins that are genomically distant from the genetically linked Isd locus. NEAT domains are protein modules that partake in several functions related to heme transport, including binding heme and hemoglobin. This finding raises interesting questions concerning the relative role of these NEAT proteins, relative to hemophores and the Isd system, in iron uptake. Here, we present evidence that a B. anthracis S-layer homology (SLH) protein harboring a NEAT domain binds and directionally transfers heme to the Isd system via the cell wall protein IsdC. This finding suggests that the Isd system can receive heme from multiple inputs and may reflect an adaptation of B. anthracis to changing iron reservoirs during an infection. Understanding the mechanism of heme uptake in pathogenic bacteria is important for the development of novel therapeutics to prevent and treat bacterial infections.Pathogenic bacteria need to acquire iron to survive in mammalian hosts (12). However, the host sequesters most iron in the porphyrin heme, and heme itself is often bound to proteins such as hemoglobin (14, 28, 85). Circulating hemoglobin can serve as a source of heme-iron for replicating bacteria in infected hosts, but the precise mechanisms of heme extraction, transport, and assimilation remain unclear (25, 46, 79, 86). An understanding of how bacterial pathogens import heme will lead to the development of new anti-infectives that inhibit heme uptake, thereby preventing or treating infections caused by these bacteria (47, 68).The mechanisms of transport of biological molecules into a bacterial cell are influenced by the compositional, structural, and topological makeup of the cell envelope. Gram-negative bacteria utilize specific proteins to transport heme through the outer membrane, periplasm, and inner membrane (83, 84). Instead of an outer membrane and periplasm, Gram-positive bacteria contain a thick cell wall (59, 60). Proteins covalently anchored to the cell wall provide a functional link between extracellular heme reservoirs and intracellular iron utilization pathways (46). In addition, several Gram-positive and Gram-negative bacterial genera also contain an outermost structure termed the S (surface)-layer (75). The S-layer is a crystalline array of protein that surrounds the bacterial cell and may serve a multitude of functions, including maintenance of cell architecture and protection from host immune components (6, 7, 18, 19, 56). In bacterial pathogens that manifest an S-layer, the “force field” function of this structure raises questions concerning how small molecules such as heme can be successfully passed from the extracellular milieu to cell wall proteins for delivery into the cell cytoplasm.Bacillus anthracis is a Gram-positive, spore-forming bacterium that is the etiological agent of anthrax disease (30, 33). The life cycle of B. anthracis begins after a phagocytosed spore germinates into a vegetative cell inside a mammalian host (2, 40, 69, 78). Virulence determinants produced by the vegetative cells facilitate bacterial growth, dissemination to major organ systems, and eventually host death (76-78). The release of aerosolized spores into areas with large concentrations of people is a serious public health concern (30).Heme acquisition in B. anthracis is mediated by the action of IsdX1 and IsdX2, two extracellular hemophores that extract heme from host hemoglobin and deliver the iron-porphyrin to cell wall-localized IsdC (21, 45). Both IsdX1 and IsdX2 harbor near iron transporter domains (NEATs), a conserved protein module found in Gram-positive bacteria that mediates heme uptake from hemoglobin and contributes to bacterial pathogenesis upon infection (3, 8, 21, 31, 44, 46, 49, 50, 67, 81, 86). Hypothesizing that B. anthracis may contain additional mechanisms for heme transport, we provide evidence that B. anthracis S-layer protein K (BslK), an S-layer homology (SLH) and NEAT protein (32, 43), is surface localized and binds and transfers heme to IsdC in a rapid, contact-dependent manner. These results suggest that the Isd system is not a self-contained conduit for heme trafficking and imply that there is functional cross talk between differentially localized NEAT proteins to promote heme uptake during infection.