The Trw Type IV Secretion System of Bartonella Mediates Host-Specific Adhesion to Erythrocytes

Bacterial pathogens typically infect only a limited range of hosts; however, the genetic mechanisms governing host-specificity are poorly understood. The α-proteobacterial genus Bartonella comprises 21 species that cause host-specific intraerythrocytic bacteremia as hallmark of infection in their respective mammalian reservoirs, including the human-specific pathogens Bartonella quintana and Bartonella bacilliformis that cause trench fever and Oroya fever, respectively. Here, we have identified bacterial factors that mediate host-specific erythrocyte colonization in the mammalian reservoirs. Using mouse-specific Bartonella birtlesii, human-specific Bartonella quintana, cat-specific Bartonella henselae and rat-specific Bartonella tribocorum, we established in vitro adhesion and invasion assays with isolated erythrocytes that fully reproduce the host-specificity of erythrocyte infection as observed in vivo. By signature-tagged mutagenesis of B. birtlesii and mutant selection in a mouse infection model we identified mutants impaired in establishing intraerythrocytic bacteremia. Among 45 abacteremic mutants, five failed to adhere to and invade mouse erythrocytes in vitro. The corresponding genes encode components of the type IV secretion system (T4SS) Trw, demonstrating that this virulence factor laterally acquired by the Bartonella lineage is directly involved in adherence to erythrocytes. Strikingly, ectopic expression of Trw of rat-specific B. tribocorum in cat-specific B. henselae or human-specific B. quintana expanded their host range for erythrocyte infection to rat, demonstrating that Trw mediates host-specific erythrocyte infection. A molecular evolutionary analysis of the trw locus further indicated that the variable, surface-located TrwL and TrwJ might represent the T4SS components that determine host-specificity of erythrocyte parasitism. In conclusion, we show that the laterally acquired Trw T4SS diversified in the Bartonella lineage to facilitate host-restricted adhesion to erythrocytes in a wide range of mammals.     

Identification of the Feline Humoral Immune Response to Bartonella henselae Infection by Protein Microarray

Background

Bartonella henselae is the zoonotic agent of cat scratch disease and causes potentially fatal infections in immunocompromised patients. Understanding the complex interactions between the host''s immune system and bacterial pathogens is central to the field of infectious diseases and to the development of effective diagnostics and vaccines.

Methodology

We report the development of a microarray comprised of proteins expressed from 96% (1433/1493) of the predicted ORFs encoded by the genome of the zoonotic pathogen Bartonella henselae. The array was probed with a collection of 62 uninfected, 62 infected, and 8 “specific-pathogen free” naïve cat sera, to profile the antibody repertoire elicited during natural Bartonella henselae infection.

Conclusions

We found that 7.3% of the B. henselae proteins on the microarray were seroreactive and that seroreactivity was not evenly distributed between predicted protein function or subcellular localization. Membrane proteins were significantly most likely to be seroreactive, although only 23% of the membrane proteins were reactive. Conversely, we found that proteins involved in amino acid transport and metabolism were significantly underrepresented and did not contain any seroreactive antigens. Of all seroreactive antigens, 52 were differentially reactive with sera from infected cats, and 53 were equally reactive with sera from infected and uninfected cats. Thirteen of the seroreactive antigens were found to be differentially seroreactive between B. henselae type I and type II. Based on these results, we developed a classifier algorithm that was capable of accurately discerning 93% of the infected animals using the microarray platform. The seroreactivity and diagnostic potential of these antigens was then validated on an immunostrip platform, which correctly identified 98% of the infected cats. Our protein microarray platform provides a high-throughput, comprehensive analysis of the feline humoral immune response to natural infection with the alpha-proteobacterium B. henselae at an antigen-specific, sera-specific, and genome-wide level. Furthermore, these results provide novel insight and utility in diagnostics, vaccine development, and understanding of host-pathogen interaction.     

Proteomic analysis of the bacterial pathogen Bartonella henselae and identification of immunogenic proteins for serodiagnosis

Bartonella henselae is a slow growing, fastidious and facultative intracellular pathogen causing cat scratch disease and vasculoproliferative disorders. To date, knowledge about the pathogenicity of this human pathogenic bacterium is limited and, additionally, serodiagnosis still needs further improvement. Here, we investigated the proteome of B. henselae using 2‐D SDS‐PAGE and MALDI‐TOF‐MS. We provide a comprehensive 2‐D proteome reference map of the whole cell lysate of B. henselae with 431 identified protein spots representing 191 different proteins of which 16 were formerly assigned as hypothetical proteins. To unravel immunoreactive antigens, we applied 2‐D SDS‐PAGE and subsequent immunoblotting using 33 sera of patients suffering from B. henselae infections. The analysis revealed 79 immunoreactive proteins of which 71 were identified. Setting a threshold of 20% seroreactivity, 11 proteins turned out to be immunodominant antigens potentially useful for an improved Bartonella‐specific serodiagnosis. Therefore, we provide for the first time (i) a comprehensive 2‐D proteome map of B. henselae for further proteome‐based studies focussed on the pathogenicity of B. henselae and (ii) an integrated view into the humoral immune responses targeted against this newly emerged human pathogenic bacterium.     

Assessment of Persistence of Bartonella henselae in Ctenocephalides felis

Bartonella henselae (Rhizobiales: Bartonellaceae) is a Gram-negative fastidious bacterium of veterinary and zoonotic importance. The cat flea Ctenocephalides felis (Siphonaptera: Pulicidae) is the main recognized vector of B. henselae, and transmission among cats and humans occurs mainly through infected flea feces. The present study documents the use of a quantitative molecular approach to follow the daily kinetics of B. henselae within the cat flea and its excreted feces after exposure to infected blood for 48 h in an artificial membrane system. B. henselae DNA was detected in both fleas and feces for the entire life span of the fleas (i.e., 12 days) starting from 24 h after initiation of the blood meal.     

Bartonella species in fleas from Palestinian territories: Prevalence and genetic diversity

Bartonellosis is an infectious bacterial disease. The prevalence and genetic characteristics of Bartonella spp. in fleas of wild and domestic animals from Palestinian territories are described. Flea samples (n=289) were collected from 121 cats, 135 dogs, 26 hyraxes and seven rats from northern (n=165), central (n=113), and southern Palestinian territories (n=11). The prevalent flea species were: Ctenocephalides felis (n=119/289; 41.2%), Ctenocephalides canis (n=159/289; 55%), and Xenopsylla sp. (n=7/289; 2.4%). Targeting the Intergenic Transcribed Spacer (ITS) locus, DNA of Bartonella was detected in 22% (64/289) of all fleas. Fifty percent of the C. felis and 57% of the Xenopsylla sp. contained Bartonella DNA. DNA sequencing showed the presence of Bartonella clarridgeiae (50%), Bartonella henselae (27%), and Bartonella koehlerae (3%) in C. felis. Xenopsylla sp. collected from Rattus rattus rats were infected with Bartonella tribocorum, Bartonella elizabethae, and Bartonella rochalimae. Phylogenetic sequence analysis using the 16S ribosomal RNA gene obtained four genetic clusters, B. henselae and B. koehlerae as subcluster 1, B. clarridgeiae as cluster 2, while the rat Bartonella species (B. tribocorum and B. elizabethae) were an outgroup cluster. These findings showed the important role of cat and rat fleas as vectors of zoonotic Bartonella species in Palestinian territories. It is hoped that this publication will raise awareness among physicians, veterinarians, and other health workers of the high prevalence of Bartonella spp. in fleas in Palestinian territories and the potential risk of these pathogens to humans and animals in this region.     

Heterologous Expression of Bartonella Adhesin A in Escherichia coli by Exchange of Trimeric Autotransporter Adhesin Domains Results in Enhanced Adhesion Properties and a Pathogenic Phenotype

Human-pathogenic Bartonella henselae causes cat scratch disease and vasculoproliferative disorders. An important pathogenicity factor of B. henselae is the trimeric autotransporter adhesin (TAA) Bartonella adhesin A (BadA), which is modularly constructed, consisting of a head, a long and repetitive neck-stalk module, and a membrane anchor. BadA is involved in bacterial autoagglutination, binding to extracellular matrix proteins and host cells, and in proangiogenic reprogramming. The slow growth of B. henselae and limited tools for genetic manipulation are obstacles for detailed examination of BadA and its domains. Here, we established a recombinant expression system for BadA mutants in Escherichia coli allowing functional analysis of particular BadA domains. Using a BadA mutant lacking 21 neck-stalk repeats (BadA HN23), the BadA HN23 signal sequence was exchanged with that of E. coli OmpA, and the BadA membrane anchor was additionally replaced with that of Yersinia adhesin A (YadA). Constructs were cloned in E. coli, and hybrid protein expression was detected by immunoblotting, fluorescence microscopy, and flow cytometry. Functional analysis revealed that BadA hybrid proteins mediate autoagglutination and binding to collagen and endothelial cells. In vivo, expression of this BadA construct correlated with higher pathogenicity of E. coli in a Galleria mellonella infection model.     

Involvement of host cell tyrosine phosphorylation in the invasion of HEp-2 cells by Bartonella bacilliformis

We have provided evidence that exposure of human cells to protein kinase inhibitors results in decreased invasion of these cells by Bartonella bacilliformis in a dose-dependent manner. Preincubation of human laryngeal epithelial cells in the presence of genistein, a tyrosine protein kinase inhibitor, decreased the invasion of these cells by B. bacilliformis significantly. Further, exposure of normal human umbilical vein endothelial cells to staurosporine, a potent inhibitor of protein kinase C and some tyrosine protein kinases, resulted in a considerable reduction in the number of organisms internalized by these cells. Moreover, Bartonella infection of HEp-2 cells induced tyrosine phosphorylation of several Triton X-100 soluble proteins with approximate molecular masses of 243, 215 179, 172 (doublet), 160, 145 and 110 kDa that were absent or reduced in the presence of genistein in cells after 1 h of infection. Exposure of HEp-2 cell monolayers to anti-alpha 5 and anti-beta 1 chain integrin monoclonal antibodies resulted in a moderate decrease in the invasion of these cells, suggesting a possible role of alpha 5 beta 1 integrins in the uptake of Bartonella into nucleated cells.     

Heme Binding Proteins of Bartonella henselae Are Required when Undergoing Oxidative Stress During Cell and Flea Invasion

Bartonella are hemotropic bacteria responsible for emerging zoonoses. These heme auxotroph alphaproteobacteria must import heme for their growth, since they cannot synthesize it. To import exogenous heme, Bartonella genomes encode for a complete heme uptake system enabling transportation of this compound into the cytoplasm and degrading it to release iron. In addition, these bacteria encode for four or five outer membrane heme binding proteins (Hbps). The structural genes of these highly homologous proteins are expressed differently depending on oxygen, temperature and heme concentrations. These proteins were hypothesized as being involved in various cellular processes according to their ability to bind heme and their regulation profile. In this report, we investigated the roles of the four Hbps of Bartonella henselae, responsible for cat scratch disease. We show that Hbps can bind heme in vitro. They are able to enhance the efficiency of heme uptake when co-expressed with a heme transporter in Escherichia coli. Using B. henselae Hbp knockdown mutants, we show that these proteins are involved in defense against the oxidative stress, colonization of human endothelial cell and survival in the flea.     

Growth Characteristics of Bartonella henselae in a Novel Liquid Medium: Primary Isolation, Growth-Phase-Dependent Phage Induction, and Metabolic Studies

Bartonella henselae is a zoonotic pathogen that usually causes a self-limiting infection in immunocompetent individuals but often causes potentially life-threatening infections, such as bacillary angiomatosis, in immunocompromised patients. Both diagnosis of infection and research into the molecular mechanisms of pathogenesis have been hindered by the absence of a suitable liquid growth medium. It has been difficult to isolate B. henselae directly from the blood of infected humans or animals or to grow the bacteria in liquid culture media under laboratory conditions. Therefore, we have developed a liquid growth medium that supports reproducible in vitro growth (3-h doubling time and a growth yield of approximately 5 × 10⁸ CFU/ml) and permits the isolation of B. henselae from the blood of infected cats. During the development of this medium, we observed that B. henselae did not derive carbon and energy from the catabolism of glucose, which is consistent with genome nucleotide sequence data suggesting an incomplete glycolytic pathway. Of interest, B. henselae depleted amino acids from the culture medium and accumulated ammonia in the medium, an indicator of amino acid catabolism. Analysis of the culture medium throughout the growth cycle revealed that oxygen was consumed and carbon dioxide was generated, suggesting that amino acids were catabolized in a tricarboxylic acid (TCA) cycle-dependent mechanism. Additionally, phage particles were detected in the culture supernatants of stationary-phase B. henselae, but not in mid-logarithmic-phase culture supernatants. Enzymatic assays of whole-cell lysates revealed that B. henselae has a complete TCA cycle. Taken together, these data suggest B. henselae may catabolize amino acids but not glucose to derive carbon and energy from its host. Furthermore, the newly developed culture medium should improve isolation of B. henselae and basic research into the pathogenesis of the bacterium.     

Detection and identification of Bartonella sp. in fleas from carnivorous mammals in Andalusia,Spain

A total of 559 fleas representing four species (Pulex irritans, Ctenocephalides felis, Ctenocephalides canis and Spilopsyllus cuniculi) collected on carnivores (five Iberian lynx Lynx pardinus, six European wildcat Felis silvestris, 10 common genet Genetta genetta, three Eurasian badger Meles meles, 22 red fox Vulpes vulpes, 87 dogs and 23 cats) in Andalusia, southern Spain, were distributed in 156 pools of monospecific flea from each carnivore, and tested for Bartonella infection in an assay based on polymerase chain reaction (PCR) amplification of the 16 S–23 S rRNA intergenic spacer region. Twenty‐one samples (13.5%) were positive and the sequence data showed the presence of four different Bartonella species. Bartonella henselae was detected in nine pools of Ctenocephalides felis from cats and dogs and in three pools of Ctenocephalides canis from cats; Bartonella clarridgeiae in Ctenocephalides felis from a cat, and Bartonella alsatica in Spilopsyllus cuniculi from a wildcat. DNA of Bartonella sp., closely related to Bartonella rochalimae, was found in seven pools of Pulex irritans from foxes. This is the first detection of B. alsatica and Bartonella sp. in the Iberian Peninsula. All of these Bartonella species have been implicated as agents of human diseases. The present survey confirms that carnivores are major reservoirs for Bartonella spp.     

Contact-dependent hemolytic activity distinct from deforming activity of Bartonella bacilliformis

Although Bartonella bacilliformis causes a severe anemia in humans, this study presents the first report of hemolytic activity by B. bacilliformis. The activity was not apparent in culture supernatants but was reliably detected when B. bacilliformis cells were centrifuged onto erythrocytes prior to incubation. Abrogation of hemolytic activity by proteinase K treatment suggested the hemolysin was a Bartonella protein. Even though hemolysis required relatively long incubation times, de novo protein synthesis was not required to produce the protein. A preparation containing factors released by B. bacilliformis, including deformin, a B. bacilliformis protein able to induce pits and invaginations in erythrocyte membranes, had some ability to lyse erythrocytes. However, pre-deformed erythrocytes did not lyse faster or to a greater extent than control erythrocytes after the addition of B. bacilliformis cells. Inhibition of deformation caused by B. bacilliformis cells with the erythrocyte ATPase inhibitor, vanadate, did not affect hemolytic activity. This study suggests hemolytic activity and deforming activity are attributable to different B. bacilliformis proteins.     

Bartonellosis,an increasingly recognized zoonosis

Cat scratch disease is the most common zoonotic infection caused by Bartonella bacteria. Among the many mammals infected with Bartonella spp., cats represent a large reservoir for human infection, as they are the main reservoir for Bartonella henselae, Bartonella clarridgeiae and Bartonella koehlerae. Bartonella spp. are vector‐borne bacteria, and transmission of B. henselae by cat fleas occurs mainly through infected flea faeces, although new potential vectors (ticks and biting flies) have been identified. Dogs are also infected with various Bartonella species and share with humans many of the clinical signs induced by these infections. Although the role of dogs as source of human infection is not yet clearly established, they represent epidemiological sentinels for human exposure. Present knowledge on the aetiology, clinical features and epidemiological characteristics of bartonellosis is presented.     

Bartonella henselae in eastern Poland: the relationship between tick infection rates and the serological response of individuals occupationally exposed to tick bites

To explore the potential role of Ixodes ricinus as the presumed vector of Bartonella henselae in eastern Poland, ticks collected in various geographic locations were examined for the presence of B. henselae, and the results were matched against the prevalence of anti‐B. henselae antibodies in individuals occupationally exposed to tick bites. The presence of Bartonella DNA was investigated by PCR in a total of 1,603 unfed Ixodes ricinus ticks. The presence of IgG antibodies against B. henselae was investigated in serum samples from 332 people occupationally exposed to tick bites (94 farmers and 238 forestry workers). The total prevalence of B. henselae in ticks was 1.7%; the infection rates in males (3.1%) and females (2.7%) were nearly ten times greater than in nymphs (0.3%). The prevalence of seropositive results in the risk group (30.4%), farmers (27.7%) and forestry workers (31.5%), was significantly greater compared to the control group (8.9%). The results showed a weak positive correlation between the degree of infection of ticks and humans living in the same geographic region. The lack of a direct relationship indicates that exposure to tick bites is only one of the factors contributing to the significant preponderance of a seropositive response to B. henselae in the forestry workers and farmers over the control group. Other factors must be considered, such as contact with cats, which are popular domestic animals in Polish villages, and exposure to cat fleas.     

Bacteriophage-like particle of Rochalimaea henselae

An extracellular particle approximately 40 nM in diameter was detected in culture supernatant from the fastidious bacterium Rochalimaea henselae. This particle has at least three associated proteins and contains 14kbp linear DNA segments that are heterogeneous in sequence. The 14kbp DNA was also present in R. henselae cells as an extrachromosomal element for all 14 strains tested. Despite attempts to induce lysis of R henselae, plaque formation was not observed. A similar particle, also containing 14 kbp DNA, was observed in Bartonella bacilliformis, and may be analogous to a bacteriophage that has been described elsewhere for B. bacilliformis.     

Prevalence of Rickettsia and Bartonella species in Spanish cats and their fleas

The aim of this study was to determine the prevalence of Bartonella henselae, Rickettsia felis, and Rickettsia typhi in fleas and companion cats (serum and claws) and to assess their presence as a function of host, host habitat, and level of parasitism. Eighty‐nine serum and claw samples and 90 flea pools were collected. Cat sera were assayed by IFA for Bartonella henselae and Rickettssia species IgG antibodies. Conventional PCRs were performed on DNA extracted from nails and fleas collected from cats. A large portion (55.8%) of the feline population sampled was exposed to at least one of the three tested vector‐borne pathogens. Seroreactivity to B. henselae was found in 50% of the feline studied population, and to R. felis in 16.3%. R. typhi antibodies were not found in any cat. No Bartonella sp. DNA was amplified from the claws. Flea samples from 41 cats (46%) showed molecular evidence for at least one pathogen; our study demonstrated a prevalence rate of 43.3 % of Rickettsia sp and 4.4% of Bartonella sp. in the studied flea population. None of the risk factors studied (cat's features, host habitat, and level of parasitation) was associated with either the serology or the PCR results for Bartonella sp. and Rickettsia sp.. Flea‐associated infectious agents are common in cats and fleas and support the recommendation that stringent flea control should be maintained on cats.     

Bartonella henselae: Subversion of vascular endothelial cell functions by translocated bacterial effector proteins

Bartonella henselae (Bh) is a worldwide distributed zoonotic pathogen. Depending on the immune status of the infected individual this bacterium can cause a wide spectrum of clinical manifestations, ranging from cat scratch disease (CSD) to bacillary angiomatosis (BA) and bacillary peliosis (BP). BA and BP are characterized by tumor-like lesions at the skin or in the inner organs, respectively. These structures display pathological sprouting of capillaries with enlarged and hyperproliferated vascular endothelial cells (ECs) that are frequently found in close association with bacteria. Here we review the cellular changes observed upon Bh infection of ECs in vitro and outline the role of the VirB type IV secretion system (T4SS) and its translocated effector proteins in the modulation of EC signalling cascades. The current model how this virulence system could contribute to the vasoproliferative activity of Bh is described.     

Bartonella spp. Bacteremia in Blood Donors from Campinas,Brazil

Bartonella species are blood-borne, re-emerging organisms, capable of causing prolonged infection with diverse disease manifestations, from asymptomatic bacteremia to chronic debilitating disease and death. This pathogen can survive for over a month in stored blood. However, its prevalence among blood donors is unknown, and screening of blood supplies for this pathogen is not routinely performed. We investigated Bartonella spp. prevalence in 500 blood donors from Campinas, Brazil, based on a cross-sectional design. Blood samples were inoculated into an enrichment liquid growth medium and sub-inoculated onto blood agar. Liquid culture samples and Gram-negative isolates were tested using a genus specific ITS PCR with amplicons sequenced for species identification. Bartonella henselae and Bartonella quintana antibodies were assayed by indirect immunofluorescence. B. henselae was isolated from six donors (1.2%). Sixteen donors (3.2%) were Bartonella-PCR positive after culture in liquid or on solid media, with 15 donors infected with B. henselae and one donor infected with Bartonella clarridgeiae. Antibodies against B. henselae or B. quintana were found in 16% and 32% of 500 blood donors, respectively. Serology was not associated with infection, with only three of 16 Bartonella-infected subjects seropositive for B. henselae or B. quintana. Bartonella DNA was present in the bloodstream of approximately one out of 30 donors from a major blood bank in South America. Negative serology does not rule out Bartonella spp. infection in healthy subjects. Using a combination of liquid and solid cultures, PCR, and DNA sequencing, this study documents for the first time that Bartonella spp. bacteremia occurs in asymptomatic blood donors. Our findings support further evaluation of Bartonella spp. transmission which can occur through blood transfusions.     

Multi-Locus Sequence Typing of a Geographically and Temporally Diverse Sample of the Highly Clonal Human Pathogen Bartonella quintana

Bartonella quintana is a re-emerging pathogen and the causative agent of a variety of disease manifestations in humans including trench fever. Various typing methods have been developed for B. quintana, but these tend to be limited by poor resolution and, in the case of gel-based methods, a lack of portability. Multilocus sequence typing (MLST) has been used to study the molecular epidemiology of a large number of pathogens, including B. henselae, a close relative of B. quintana. We developed a MLST scheme for B. quintana based on the 7 MLST loci employed for B. henselae with two additional loci to cover underrepresented regions of the B. quintana chromosome. A total of 16 B. quintana isolates spanning over 60 years and three continents were characterized. Allelic variation was detected in five of the nine loci. Although only 8/4270 (0.002%) of the nucleotide sites examined were variable over all loci, these polymorphisms resolved the 16 isolates into seven sequence types (STs). We also demonstrate that MLST can be applied on uncultured isolates by direct PCR from cardiac valve tissue, and suggest this method presents a promising approach for epidemiological studies in this highly clonal organism. Phylogenetic and clustering analyses suggest that two of the seven STs form a distinct lineage within the population.     

Gene duplication and deletion,not horizontal transfer,drove intra-species mosaicism of Bartonella henselae

Bartonella henselae is a facultative intracellular pathogen that occurs worldwide and is responsible primarily for cat-scratch disease in young people and bacillary angiomatosis in immunocompromised patients. The principal source of genome-level diversity that contributes to B. henselae's host-adaptive features is thought to be horizontal gene transfer events. However, our analyses did not reveal the acquisition of horizontally-transferred islands in B. henselae after its divergence from other Bartonella. Rather, diversity in gene content and genome size was apparently acquired through two alternative mechanisms, including deletion and, more predominantly, duplication of genes. Interestingly, a majority of these events occurred in regions that were horizontally transferred long before B. henselae's divergence from other Bartonella species. Our study indicates the possibility that gene duplication, in response to positive selection pressures in specific clones of B. henselae, might be linked to the pathogen's adaptation to arthropod vectors, the cat reservoir, or humans as incidental host-species.