The autotransporter protein from Bordetella avium,Baa1, is involved in host cell attachment

Bordetella avium is a Gram negative upper respiratory tract pathogen of birds. B. avium infection of commercially raised turkeys is an agriculturally significant problem. Here we describe the functional analysis of the first characterized B. avium autotransporter protein, Baa1. Autotransporters comprise a large family of proteins found in all groups of Gram negative bacteria. Although not unique to pathogenic bacteria, autotransporters have been shown to perform a variety of functions implicated in virulence. To test the hypothesis that Baa1 is a B. avium virulence factor, unmarked baa1 deletion mutants (Δbaa1) were created and tested phenotypically. It was found that baa1 mutants have wild-type levels of serum sensitivity and infectivity, yet significantly lower levels of turkey tracheal cell attachment in vitro. Likewise, semi-purified recombinant His-tagged Baa1, expressed in Escherichia coli, was shown to bind specifically to turkey tracheal cells via western blot analysis. Taken together, we conclude that Baa1 acts as a host cell attachment factor and thus plays a role B. avium virulence.     

Design,synthesis and in vivo/in vitro screening of novel chlorokojic acid derivatives

A series of novel Mannich bases of chlorokojic acid (2-chloromethyl-5-hydroxy-4H-pyran-4-one) were synthesized and their biological activities were investigated. Anticonvulsant activity results according to phase-I tests of Antiepileptic Drug Development (ADD) Program revealed that compound 13 was the most effective one at 4?h against subcutaneous pentylenetetrazole (scPTZ)-induced seizure test. Antimicrobial activities were evaluated in vitro against bacteria and fungi by using broth microdilution method. The antitubercular activities against Mycobacterium tuberculosis and M. avium were discussed with Resazurin microplate assay (REMA). The antimicrobial activity results indicated that compounds 1 and 12 (MIC: 8–16 µg/mL) showed higher activity against Gram negative bacteria while compound 12 had MIC: 4–16 µg/mL against Gram positive bacteria. Compound 1 was the most active one with MIC values of 8–32 µg/mL against fungi. Mannich bases also exhibit significant antitubercular activity in a MIC range of 4 to 32 µg/mL, especially compound 18 against M. avium.     

Structure of the bacterial cell division determinant GpsB and its interaction with penicillin‐binding proteins

Each bacterium has to co‐ordinate its growth with division to ensure genetic stability of the population. Consequently, cell division and growth are tightly regulated phenomena, albeit different bacteria utilise one of several alternative regulatory mechanisms to maintain control. Here we consider GpsB, which is linked to cell growth and division in Gram‐positive bacteria. ΔgpsB mutants of the human pathogen Listeria monocytogenes show severe lysis, division and growth defects due to distortions of cell wall biosynthesis. Consistent with this premise, GpsB interacts both in vitro and in vivo with the major bi‐functional penicillin‐binding protein. We solved the crystal structure of GpsB and the interaction interfaces in both proteins are identified and validated. The inactivation of gpsB results in strongly attenuated virulence in animal experiments, comparable in degree to classical listerial virulence factor mutants. Therefore, GpsB is essential for in vitro and in vivo growth of a highly virulent food‐borne pathogen, suggesting that GpsB could be a target for the future design of novel antibacterials.     

Cyclic diguanylate regulation of Bacillus cereus group biofilm formation

Biofilm formation can be considered a bacterial virulence mechanism. In a range of Gram‐negatives, increased levels of the second messenger cyclic diguanylate (c‐di‐GMP) promotes biofilm formation and reduces motility. Other bacterial processes known to be regulated by c‐di‐GMP include cell division, differentiation and virulence. Among Gram‐positive bacteria, where the function of c‐di‐GMP signalling is less well characterized, c‐di‐GMP was reported to regulate swarming motility in Bacillus subtilis while having very limited or no effect on biofilm formation. In contrast, we show that in the Bacillus cereus group c‐di‐GMP signalling is linked to biofilm formation, and to several other phenotypes important to the lifestyle of these bacteria. The Bacillus thuringiensis 407 genome encodes eleven predicted proteins containing domains (GGDEF/EAL) related to c‐di‐GMP synthesis or breakdown, ten of which are conserved through the majority of clades of the B. cereus group, including Bacillus anthracis. Several of the genes were shown to affect biofilm formation, motility, enterotoxin synthesis and/or sporulation. Among these, cdgF appeared to encode a master diguanylate cyclase essential for biofilm formation in an oxygenated environment. Only two cdg genes (cdgA, cdgJ) had orthologs in B. subtilis, highlighting differences in c‐di‐GMP signalling between B. subtilis and B. cereus group bacteria.     

Restoration of attachment, virulence and nodulation of Agrobacterium tumefaciens chvB mutants by rhicadhesin

In contrast to wild-type Agrobacterium tumefaciens strains, β-1,2-glucan-deficient chvB mutants were found to be unable to attach to pea root hair tips. The mutants appeared to produce rhicadhesin, the protein that mediates the first step in attachment of Rhizobiaceae cells to plant root hairs, but the protein was inactive. Both attachment to root hairs and virulence of the ChvB mutants could be restored by treatment of the plants with active rhicadhesin, whereas treatment of plants with β-1,2-glucan had no effect on attachment or virulence. Moreover, nodulation ability of a chvB mutant carrying a Sym plasmid could be restored by pretreatment of the host plant with rhicadhesin. Apparently the attachment-minus and avirulence phenotype of chvB mutants is caused by lack of active rhicadhesin, rather than directly being caused by a deficiency in β-1,2-glucan synthesis. The results strongly suggest that rhicadhesin is essential for attachment and virulence of A. tumefaciens cells. They also indicate that the mechanisms of binding of Agrobacterium and Rhizobium bacteria to plant target cells are similar, despite differences between these target cells.     

Bacterial membrane vesicles deliver peptidoglycan to NOD1 in epithelial cells

Gram‐negative bacterial peptidoglycan is specifically recognized by the host intracellular sensor NOD1, resulting in the generation of innate immune responses. Although epithelial cells are normally refractory to external stimulation with peptidoglycan, these cells have been shown to respond in a NOD1‐dependent manner to Gram‐negative pathogens that can either invade or secrete factors into host cells. In the present work, we report that Gram‐negative bacteria can deliver peptidoglycan to cytosolic NOD1 in host cells via a novel mechanism involving outer membrane vesicles (OMVs). We purified OMVs from the Gram‐negative mucosal pathogens: Helicobacter pylori, Pseudomonas aeruginosa and Neisseria gonorrhoea and demonstrated that these peptidoglycan containing OMVs upregulated NF‐κB and NOD1‐dependent responses in vitro. These OMVs entered epithelial cells through lipid rafts thereby inducing NOD1‐dependent responses in vitro. Moreover, OMVs delivered intragastrically to mice‐induced innate and adaptive immune responses via a NOD1‐dependent but TLR‐independent mechanism. Collectively, our findings identify OMVs as a generalized mechanism whereby Gram‐negative bacteria deliver peptidoglycan to cytosolic NOD1. We propose that OMVs released by bacteria in vivo may promote inflammation and pathology in infected hosts.     

Evaluation of an in vitro cell assay to select attenuated bacterial mutants of Aeromonas hydrophila and Edwardsiella tarda to channel catfish

Aims: To evaluate the feasibility of using an in vitro cell assay to select attenuated bacterial mutants. Methods and Results: Using catfish gill cells G1B, the feasibility of using an in vitro assay instead of in vivo virulence assay using live fish to select attenuated bacterial mutants was evaluated in this study. Pearson correlation analysis between in vitro virulence to G1B cells and in vivo virulence of Aeromonas hydrophila and Edwardsiella tarda revealed that there was a significant correlation between the two (r = ?0·768, P value = 3·7 × 10^?16). Conclusions: The in vitro cell assay might be initially used to screen large quantities of bacteria to select attenuated mutants of catfish pathogens. Significance and Impact of the Study: The in vitro cell assay using catfish gill cells to identify attenuated mutants of catfish pathogens will reduce cost involved in the in vivo virulence assay that requires many fish and aquariums.     

Bordetella avium causes induction of apoptosis and nitric oxide synthase in turkey tracheal explant cultures

Bordetellosis is an upper respiratory disease of turkeys caused by Bordetella avium in which the bacteria attach specifically to ciliated respiratory epithelial cells. Little is known about the mechanisms of pathogenesis of this disease, which has a negative impact in the commercial turkey industry. In this study, we produced a novel explant organ culture system that was able to successfully reproduce pathogenesis of B. avium in vitro, using tracheal tissue derived from 26 day-old turkey embryos. Treatment of the explants with whole cells of B. avium virulent strain 197N and culture supernatant, but not lipopolysaccharide (LPS) or tracheal cytotoxin (TCT), specifically induced apoptosis in ciliated cells, as shown by annexin V and TUNEL staining. LPS and TCT are known virulence factors of Bordetella pertussis, the causative agent of whooping cough. Treatment with whole cells of B. avium and LPS specifically induced NO response in ciliated cells, shown by uNOS staining and diaphorase activity. The explant system is being used as a model to elucidate specific molecules responsible for the symptoms of bordetellosis.     

Streptococcus mutans requires mature rhamnose‐glucose polysaccharides for proper pathophysiology,morphogenesis and cellular division

The cell wall of Gram‐positive bacteria has been shown to mediate environmental stress tolerance, antibiotic susceptibility, host immune evasion and overall virulence. The majority of these traits have been demonstrated for the well‐studied system of wall teichoic acid (WTA) synthesis, a common cell wall polysaccharide among Gram‐positive organisms. Streptococcus mutans, a Gram‐positive odontopathogen that contributes to the enamel‐destructive disease dental caries, lacks the capabilities to generate WTA. Instead, the cell wall of S. mutans is highly decorated with rhamnose‐glucose polysaccharides (RGP), for which functional roles are poorly defined. Here, we demonstrate that the RGP has a distinct role in protecting S. mutans from a variety of stress conditions pertinent to pathogenic capability. Mutant strains with disrupted RGP synthesis failed to properly localize cell division complexes, suffered from aberrant septum formation and exhibited enhanced cellular autolysis. Surprisingly, mutant strains of S. mutans with impairment in RGP side chain modification grew into elongated chains and also failed to properly localize the presumed cell wall hydrolase, GbpB. Our results indicate that fully mature RGP has distinct protective and morphogenic roles for S. mutans, and these structures are functionally homologous to the WTA of other Gram‐positive bacteria.     

A role for lipopolysaccharide in turkey tracheal colonization by Bordetella avium as demonstrated in vivo and in vitro

We isolated two insertion mutants of Bordetella avium that exhibited a peculiar clumped-growth phenotype and found them to be attenuated in turkey tracheal colonization. The mutants contained transposon insertions in homologues of the wlbA and wlbL genes of Bordetella pertussis. The wlb genetic locus of B. pertussis has been previously described as containing 12 genes involved in lipopolysaccharide (LPS) biosynthesis. Polyacrylamide gel analysis of LPS from B. avium wlbA and wlbL insertion mutants confirmed an alteration in the LPS profile. Subsequent cloning and complementation of the wlbA and wlbL mutants in trans with a recombinant plasmid containing the homologous wlb locus from B. avium eliminated the clumped-growth phenotype and restored the LPS profile to that of wild-type B. avium. Also, a parental level of tracheal colonization was restored to both mutants by the recombinant plasmid. Interestingly, complementation of the wlbA and wlbL mutants with a recombinant plasmid containing the heterologous wlb locus from B. pertussis, B. bronchiseptica, or Bordetella parapertussis eliminated the clumped-growth phenotype and resulted in a change in the LPS profile, although not to that of wild-type B. avium. The mutants also acquired resistance to a newly identified B. avium-specific bacteriophage, Ba1. Complementation of both wlbA and wlbL mutants with the homologous wlb locus of B. avium, but not the heterologous B. pertussis locus, restored sensitivity to Ba1. Complementation of the wlbL mutant, but not the wlbA mutant, with the heterologous wlb locus of Bordetella bronchiseptica or B. parapertussis restored partial sensitivity to Ba1. Comparisons of the LPS profile and phage sensitivity of the mutants upon complementation by wlb loci from the heterologous species and by B. avium suggested that phage sensitivity required the presence of O-antigen. At the mechanistic level, both mutants showed a dramatic decrease in serum resistance and a decrease in binding to turkey tracheal rings in vitro. In the case of serum resistance, complementation of both mutants with the homologous wlb locus of B. avium restored serum resistance to wild-type levels. However, in the case of epithelial cell binding, only complementation of the wlbA mutant completely restored binding to wild-type levels (binding was only partially restored in the wlbL mutant). This is the first characterization of LPS mutants of B. avium at the genetic level and the first report of virulence changes by both in vivo and in vitro measurements.     

NaxD is a deacetylase required for lipid A modification and Francisella pathogenesis

Modification of specific Gram‐negative bacterial cell envelope components, such as capsule, O‐antigen and lipid A, are often essential for the successful establishment of infection. Francisella species express lipid A molecules with unique characteristics involved in circumventing host defences, which significantly contribute to their virulence. In this study, we show that NaxD, a member of the highly conserved YdjC superfamily, is a deacetylase required for an important modification of the outer membrane component lipid A in Francisella. Mass spectrometry analysis revealed that NaxD is essential for the modification of a lipid A phosphate with galactosamine in Francisella novicida, a model organism for the study of highly virulent Francisella tularensis. Significantly, enzymatic assays confirmed that this protein is necessary for deacetylation of its substrate. In addition, NaxD was involved in resistance to the antimicrobial peptide polymyxin B and critical for replication in macrophages and in vivo virulence. Importantly, this protein is also required for lipid A modification in F. tularensis as well as Bordetella bronchiseptica. Since NaxD homologues are conserved among many Gram‐negative pathogens, this work has broad implications for our understanding of host subversion mechanisms of other virulent bacteria.     

Botrytis cinerea mutants deficient in d‐galacturonic acid catabolism have a perturbed virulence on Nicotiana benthamiana and Arabidopsis,but not on tomato

d ‐Galacturonic acid is the most abundant monosaccharide component of pectic polysaccharides that comprise a significant part of most plant cell walls. Therefore, it is potentially an important nutritional factor for Botrytis cinerea when it grows in and through plant cell walls. The d ‐galacturonic acid catabolic pathway in B. cinerea consists of three catalytic steps converting d ‐galacturonic acid to pyruvate and l ‐glyceraldehyde, involving two nonhomologous galacturonate reductase genes (Bcgar1 and Bcgar2), a galactonate dehydratase gene (Bclgd1) and a 2‐keto‐3‐deoxy‐l ‐galactonate aldolase gene (Bclga1). Knockout mutants in each step of the pathway (ΔBcgar1/ΔBcgar2, ΔBclgd1 and ΔBclga1) showed reduced virulence on Nicotiana benthamiana and Arabidopsis thaliana leaves, but not on Solanum lycopersicum leaves. The cell walls of N. benthamiana and A. thaliana leaves were shown to have a higher d ‐galacturonic acid content relative to those of S. lycopersicum. The observation that mutants displayed a reduction in virulence, especially on plants with a high d ‐galacturonic acid content in the cell walls, suggests that, in these hosts, d ‐galacturonic acid has an important role as a carbon nutrient for B. cinerea. However, additional in vitro growth assays with the knockout mutants revealed that B. cinerea growth is reduced when d ‐galacturonic acid catabolic intermediates cannot proceed through the entire pathway, even when fructose is present as the major, alternative carbon source. These data suggest that the reduced virulence of d ‐galacturonic acid catabolism‐deficient mutants on N. benthamiana and A. thaliana is not only a result of the inability of the mutants to utilize an abundant carbon source as nutrient, but also a result of the growth inhibition by catabolic intermediates.     

GacA is essential for Group A Streptococcus and defines a new class of monomeric dTDP‐4‐dehydrorhamnose reductases (RmlD)

The sugar nucleotide dTDP‐L‐rhamnose is critical for the biosynthesis of the Group A Carbohydrate, the molecular signature and virulence determinant of the human pathogen Group A Streptococcus (GAS). The final step of the four‐step dTDP‐L‐rhamnose biosynthesis pathway is catalyzed by dTDP‐4‐dehydrorhamnose reductases (RmlD). RmlD from the Gram‐negative bacterium Salmonella is the only structurally characterized family member and requires metal‐dependent homo‐dimerization for enzymatic activity. Using a biochemical and structural biology approach, we demonstrate that the only RmlD homologue from GAS, previously renamed GacA, functions in a novel monomeric manner. Sequence analysis of 213 Gram‐negative and Gram‐positive RmlD homologues predicts that enzymes from all Gram‐positive species lack a dimerization motif and function as monomers. The enzymatic function of GacA was confirmed through heterologous expression of gacA in a S. mutans rmlD knockout, which restored attenuated growth and aberrant cell division. Finally, analysis of a saturated mutant GAS library using Tn‐sequencing and generation of a conditional‐expression mutant identified gacA as an essential gene for GAS. In conclusion, GacA is an essential monomeric enzyme in GAS and representative of monomeric RmlD enzymes in Gram‐positive bacteria and a subset of Gram‐negative bacteria. These results will help future screens for novel inhibitors of dTDP‐L‐rhamnose biosynthesis.     

The type VI protein secretion system contributes to biofilm formation and seed‐to‐seedling transmission of Acidovorax citrulli on melon

The type VI protein secretion system (T6SS) is essential for the virulence of several Gram‐negative bacteria. In this study, we identified a T6SS gene cluster in Acidovorax citrulli, a plant‐pathogenic bacterium that causes bacterial fruit blotch (BFB) of cucurbits. One T6SS cluster, of approximately 25 kb in length and comprising 17 genes, was found in the A. citrulli AAC00‐1 genome. Seventeen A. citrulli mutants were generated, each with a deletion of a single T6SS core gene. There were significant differences in BFB seed‐to‐seedling transmission between wild‐type A. citrulli strain, xjl12, and ΔvasD, ΔimpK, ΔimpJ and ΔimpF mutants (71.71%, 9.83%, 8.41%, 7.15% and 5.99% BFB disease index, respectively). In addition, we observed that these four mutants were reduced in melon seed colonization and biofilm formation; however, they were not affected in virulence when infiltrated into melon seedling tissues. There were no significant differences in BFB seed‐to‐seedling transmission, melon tissue colonization and biofilm formation between xjl12 and the other 13 T6SS mutants. Overall, our results indicate that T6SS plays a role in seed‐to‐seedling transmission of BFB on melon.     

Attachment Strength of Listeria monocytogenes and its Internalin-Negative Mutants

A single cell of Listeria monocytogenes attached on food contact surfaces can be a potential source of cross-contamination in a food-processing plant. To see whether internalin A (InlA) and B (InlB), major surface proteins on L. monocytogenes, play a significant role in the attachment process, wild-type L. monocytogenes EGD (LM_EGD) and its isogenic internalin-negative mutants (LM_EGDΔinlA, LM_EGDΔinlB, and LM_EGDΔinlAB) were used to determine attachment strength on inert glass surface. Western blot analysis using InlA and InlB antibodies confirmed the absence of InlA in LM_EGDΔinlA, InlB in LM_EGDΔinlB, and both InlA and InlB in LM_EGDΔinlAB. Regardless of initial attachment numbers, LM_EGD which expressed both InlA and InlB proteins exhibited the strongest attachment strength while the double mutant (LM_EGDΔinlAB) exhibited the weakest. The two single mutants (LM_EGDΔinlA and LM_EGDΔinlB) that expressed only one type of the internalins were shown to have intermediate attachment strength. These results suggest that both InlA and InlB expression play a significant role in the attachment strength of L. monocytogenes on glass surface.     

Extracellular HtrA serine proteases: An emerging new strategy in bacterial pathogenesis

The HtrA family of chaperones and serine proteases is important for regulating stress responses and controlling protein quality in the periplasm of bacteria. HtrA is also associated with infectious diseases since inactivation of htrA genes results in significantly reduced virulence properties by various bacterial pathogens. These virulence features of HtrA can be attributed to reduced fitness of the bacteria, higher susceptibility to environmental stress and/or diminished secretion of virulence factors. In some Gram‐negative and Gram‐positive pathogens, HtrA itself can be exposed to the extracellular environment promoting bacterial colonisation and invasion of host tissues. Most of our knowledge on the function of exported HtrAs stems from research on Helicobacter pylori, Campylobacter jejuni, Borrelia burgdorferi, Bacillus anthracis, and Chlamydia species. Here, we discuss recent progress showing that extracellular HtrAs are able to cleave cell‐to‐cell junction factors including E‐cadherin, occludin, and claudin‐8, as well as extracellular matrix proteins such as fibronectin, aggrecan, and proteoglycans, disrupting the epithelial barrier and producing substantial host cell damage. We propose that the export of HtrAs is a newly discovered strategy, also applied by additional bacterial pathogens. Consequently, exported HtrA proteases represent highly attractive targets for antibacterial treatment by inhibiting their proteolytic activity or application in vaccine development.     

Insights into the autotransport process of a trimeric autotransporter,Yersinia Adhesin A (YadA)

Trimeric autotransporter adhesins (TAAs) are a subset of a larger protein family called the type V secretion systems. They are localized on the cell surface of Gram‐negative bacteria, function as mediators of attachment to inorganic surfaces and host cells, and thus include important virulence factors. Yersinia adhesin A (YadA) from Yersinia enterocolitica is a prototypical TAA that is used extensively to study the structure and function of the type Vc secretion system. A solid‐state NMR study of the membrane anchor domain of YadA previously revealed a flexible stretch of small residues, termed the ASSA region, that links the membrane anchor to the stalk domain. In this study, we present evidence that single amino acid proline substitutions produce two different conformers of the membrane anchor domain of YadA; one with the N‐termini facing the extracellular surface, and a second with the N‐termini located in the periplasm. We propose that TAAs adopt a hairpin intermediate during secretion, as has been shown before for other subtypes of the type V secretion system. As the YadA transition state intermediate can be isolated from the outer membrane, future structural studies should be possible to further unravel details of the autotransport process.     

Biofilm formation in an ice cream plant

The sites of biofilm formation in an ice cream plant were investigated by sampling both the production line and the environment. Experiments were carried out twice within a 20-day period. First, stainless steel coupons were fixed to surfaces adjacent to food contact surfaces, the floor drains and the doormat. They were taken for the analysis of biofilm at three different production stages. Then, biofilm forming bacteria were␣enumerated and also presence of Listeria monocytogenes was monitored. Biofilm forming isolates were selected on the basis of colony morphology and Gram’s reaction; Gram negative cocci and rod, Gram positive cocci and spore forming isolates were identified. Most of the biofilm formations were seen on the conveyor belt of a packaging machine 8 h after the beginning of the production, 6.5 × 10³ cfu cm⁻². Most of the Gram negative bacteria identified belong to Enterobacteriaceae family such as Proteus, Enterobacter, Citrobacter, Shigella, Escherichia, Edwardsiella. The other Gram negative microflora included Aeromonas, Plesiomonas, Moraxella, Pseudomonas or Alcaligenes spp. were also isolated. Gram positive microflora of the ice cream plant included Staphyloccus, Bacillus, Listeria and lactic acid bacteria such as Streptococcus, Leuconostoc or Pediococcus spp. The results from this study highlighted the problems of spread of pathogens like Listeria and Shigella and spoilage bacteria. In the development of cleaning and disinfection procedures in ice cream plants, an awareness of these biofilm-forming bacteria is essential for the ice cream plants.