Capsule Mutant Research Articles

Optimal replication and suppression of inflammation by virulent Francisella tularensis is achieved through reprogramming of host glycolysis.

Abstract Francisella tularensis (Ft) is a gram negative, facultative intracellular bacterium that is the causative agent of pneumonic tularemia. Inhalation of as a few as 15 organisms can result in lethal infection. The virulence of Ft is attributed to the bacterium’s ability to both evade and suppress the activation of macrophages. However, the mechanisms and bacterial components required for evasion and suppression by Ft are unclear. Activation of macrophages and induction of antimicrobial responses requires a metabolic shift from oxidative phosphorylation to aerobic glycolysis. Thus, we postulated that Ft manipulates host metabolism as a mechanism of virulence. We established that virulent Ft suppresses induction of aerobic glycolysis among infected macrophages. Moreover, utilizing purified capsule and defined capsule mutants, we elucidated a novel role for Ft capsule as a component contributing to this suppression. The requirement for inhibition of host cell glycolysis in Ft pathogenesis was confirmed via direct inhibition of glycolysis using 2-deoxyglucose. Addition of 2-DG to macrophages infected with capsule mutants partially restored the early intracellular survival and replication of these bacteria, and impaired the secretion of pro-inflammatory cytokines that is typical of infection with these mutants. Together, our data demonstrate that manipulation of host metabolism is an important component of Ft pathogenesis and uncovers a previously unappreciated function of bacterial capsule in the virulence of Ft.

Surface Polysaccharide Mutants Reveal that Absence of O Antigen Reduces Biofilm Formation of Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae is a Gram-negative bacterium belonging to the Pasteurellaceae family and the causative agent of porcine pleuropneumonia, a highly contagious lung disease causing important economic losses. Surface polysaccharides, including lipopolysaccharides (LPS) and capsular polysaccharides (CPS), are implicated in the adhesion and virulence of A. pleuropneumoniae, but their role in biofilm formation is still unclear. In this study, we investigated the requirement for these surface polysaccharides in biofilm formation by A. pleuropneumoniae serotype 1. Well-characterized mutants were used: an O-antigen LPS mutant, a truncated core LPS mutant with an intact O antigen, a capsule mutant, and a poly-N-acetylglucosamine (PGA) mutant. We compared the amount of biofilm produced by the parental strain and the isogenic mutants using static and dynamic systems. Compared to the findings for the biofilm of the parental or other strains, the biofilm of the O antigen and the PGA mutants was dramatically reduced, and it had less cell-associated PGA. Real-time PCR analyses revealed a significant reduction in the level of pgaA, cpxR, and cpxA mRNA in the biofilm cells of the O-antigen mutant compared to that in the biofilm cells of the parental strain. Specific binding between PGA and LPS was consistently detected by surface plasmon resonance, but the lack of O antigen did not abolish these interactions. In conclusion, the absence of the O antigen reduces the ability of A. pleuropneumoniae to form a biofilm, and this is associated with the reduced expression and production of PGA.

Type 3 secretion system cluster 3 is a critical virulence determinant for lung-specific melioidosis.

Burkholderia pseudomallei, the bacterial agent of melioidosis, causes disease through inhalation of infectious particles, and is classified as a Tier 1 Select Agent. Optical diagnostic imaging has demonstrated that murine respiratory disease models are subject to significant upper respiratory tract (URT) colonization. Because human melioidosis is not associated with URT colonization as a prominent presentation, we hypothesized that lung-specific delivery of B. pseudomallei may enhance our ability to study respiratory melioidosis in mice. We compared intranasal and intubation-mediated intratracheal (IMIT) instillation of bacteria and found that the absence of URT colonization correlates with an increased bacterial pneumonia and systemic disease progression. Comparison of the LD50 of luminescent B. pseudomallei strain, JW280, in intranasal and IMIT challenges of albino C57BL/6J mice identified a significant decrease in the LD50 using IMIT. We subsequently examined the LD50 of both capsular polysaccharide and Type 3 Secretion System cluster 3 (T3SS3) mutants by IMIT challenge of mice and found that the capsule mutant was attenuated 6.8 fold, while the T3SS3 mutant was attenuated 290 fold, demonstrating that T3SS3 is critical to respiratory melioidosis. Our previously reported intranasal challenge studies, which involve significant URT colonization, did not identify a dissemination defect for capsule mutants; however, we now report that capsule mutants exhibit significantly reduced dissemination from the lung following lung-specific instillation, suggesting that capsule mutants are competent to spread from the URT, but not the lung. We also report that a T3SS3 mutant is defective for dissemination following lung-specific delivery, and also exhibits in vivo growth defects in the lung. These findings highlight the T3SS3 as a critical virulence system for respiratory melioidosis, not only in the lung, but also for subsequent spread beyond the lung using a model system uniquely capable to characterize the fate of lung-delivered pathogen.

Klebsiella pneumoniaetargets an EGF receptor-dependent pathway to subvert inflammation

The NF-κB transcriptional factor plays a key role governing the activation of immune responses. Klebsiella pneumoniae is an important cause of community-acquired and nosocomial pneumonia. Evidence indicates that K. pneumoniae infections are characterized by lacking an early inflammatory response. Recently, we have demonstrated that Klebsiella antagonizes the activation of NF-κB via the deubiquitinase CYLD. In this work, by applying a high-throughput siRNA gain-of-function screen interrogating the human kinome, we identified 17 kinases that when targeted by siRNA restored IL-1β-dependent NF-κB translocation in infected cells. Further characterization revealed that K. pneumoniae activates an EGF receptor (EGFR)-phosphatidylinositol 3-OH kinase (PI3K)-AKT-PAK4-ERK-GSK3β signalling pathway to attenuate the cytokine-dependent nuclear translocation of NF-κB. Our data also revealed that CYLD is a downstream effector of K. pneumoniae-induced EGFR-PI3K-AKT-PAK4-ERK-GSK3β signalling pathway. Our efforts to identify the bacterial factor(s)responsible for EGFR activation demonstrate that a capsule (CPS) mutant did not activate EGFR hence suggesting that CPS could mediate the activation of EGFR. Supporting this notion, purified CPS did activate EGFR as well as the EGFR-dependent PI3K-AKT-PAK4-ERK-GSK3β signalling pathway. CPS-mediated EGFR activation was dependent on a TLR4-MyD88-c-SRC-dependent pathway. Several promising drugs have been developed to antagonize this cascade. We propose that agents targeting this signalling pathway might provide selective alternatives for the management of K. pneumoniae pneumonias.

Immunomodulatory Effects of Streptococcus suis Capsule Type on Human Dendritic Cell Responses, Phagocytosis and Intracellular Survival

Streptococcus suis is a major porcine pathogen of significant commercial importance worldwide and an emerging zoonotic pathogen of humans. Given the important sentinel role of mucosal dendritic cells and their importance in induction of T cell responses we investigated the effect of different S. suis serotype strains and an isogenic capsule mutant of serotype 2 on the maturation, activation and expression of IL-10, IL-12p70 and TNF-α in human monocyte-derived dendritic cells. Additionally, we compared phagocytosis levels and bacterial survival after internalization. The capsule of serotype 2, the most common serotype associated with infection in humans and pigs, was highly anti-phagocytic and modulated the IL-10/IL-12 and IL-10/TNF-α cytokine production in favor of a more anti-inflammatory profile compared to other serotypes. This may have consequences for the induction of effective immunity to S. suis serotype 2 in humans. A shielding effect of the capsule on innate Toll-like receptor signaling was also demonstrated. Furthermore, we showed that 24 h after phagocytosis, significant numbers of viable intracellular S. suis were still present intracellularly. This may contribute to the dissemination of S. suis in the body.

Genome Wide Expression Profiling Reveals Suppression of Host Defence Responses during Colonisation by Neisseria meningitides but not N. lactamica

Both Neisseria meningitidis and the closely related bacterium Neisseria lactamica colonise human nasopharyngeal mucosal surface, but only N. meningitidis invades the bloodstream to cause potentially life-threatening meningitis and septicaemia. We have hypothesised that the two neisserial species differentially modulate host respiratory epithelial cell gene expression reflecting their disease potential. Confluent monolayers of 16HBE14 human bronchial epithelial cells were exposed to live and/or dead N. meningitidis (including capsule and pili mutants) and N. lactamica, and their transcriptomes were compared using whole genome microarrays. Changes in expression of selected genes were subsequently validated using Q-RT-PCR and ELISAs. Live N. meningitidis and N. lactamica induced genes involved in host energy production processes suggesting that both bacterial species utilise host resources. N. meningitidis infection was associated with down-regulation of host defence genes. N. lactamica, relative to N. meningitidis, initiates up-regulation of proinflammatory genes. Bacterial secreted proteins alone induced some of the changes observed. The results suggest N. meningitidis and N. lactamica differentially regulate host respiratory epithelial cell gene expression through colonisation and/or protein secretion, and that this may contribute to subsequent clinical outcomes associated with these bacteria.

Bioluminescent diagnostic imaging to characterize altered respiratory tract colonization by the burkholderia pseudomallei capsule mutant.

Pneumonia is a common manifestation of the potentially fatal disease melioidosis, caused by the select agent bacteria Burkholderia pseudomallei. In this study we describe a new model system to investigate pulmonary melioidosis in vivo using bioluminescent-engineered bacteria in a murine respiratory disease model. Studies were performed to validate that the stable, light producing B. pseudomallei strain JW280 constitutively produced light in biologically relevant host–pathogen interactions. Hairless outbred SKH1 mice were used to enhance the ability to monitor B. pseudomallei respiratory disease, and were found to be similarly susceptible to respiratory melioidosis as BALB/c mice. This represents the first demonstration of in vivo diagnostic imaging of pulmonary melioidosis permitting the detection of B. pseudomallei less than 24 h post-infection. Diagnostic imaging of pulmonary melioidosis revealed distinct temporal patterns of bacterial colonization unique to both BALB/c and SKH1 mice. Validation of these model systems included the use of the previously characterized capsule mutant, which was found to colonize the upper respiratory tract at significantly higher levels than the wild type strain. These model systems allow for high resolution detection of bacterial pulmonary disease which will facilitate studies of therapeutics and basic science evaluation of melioidosis.

The Streptococcus pneumoniae Capsule Is Required for Full Virulence in Pneumococcal Endophthalmitis

To determine whether Streptococcus pneumoniae capsule was necessary for pathogenesis of pneumococcal endophthalmitis. An isogenic capsule-deficient strain was created using homologous recombination. New Zealand White rabbits were injected intravitreously with 10(2) colony-forming units (CFU) of the parent strain or the capsule mutant. Slit lamp examination (SLE), electroretinography, and myeloperoxidase activity were performed 24 and 48 hours postinfection (PI). Serial dilutions of vitreous were plated to quantitate CFU, eyes were extracted for histology, and host cytokine mRNA expression was determined. Eyes infected with the parent strain had significantly higher SLE scores than eyes infected with the capsule-deficient strain 24 and 48 hours PI (P < 0.001). CFU recovered from eyes infected with the capsule mutant were significantly fewer than CFU recovered from eyes infected with the parent strain 24 and 48 hours PI (P < 0.001). The parent strain caused a significantly greater decrease in retinal function and more retinal destruction than the mutant strain 48 hours PI (P = 0.026). Vitreal IL-1β, IL-6, and TNF-α were upregulated by both the parent and mutant strain 12 hours PI. By 48 hours PI, there was significantly more neutrophil infiltration in the vitreous infected with the parent strain. Endophthalmitis caused by the encapsulated strain is more damaging to retinal function and structural integrity. These findings indicate that capsule is an important virulence factor of S. pneumoniae endophthalmitis, in contrast to keratitis, suggesting that the anatomic host site in pneumococcal ocular infections is important.

Klebsiella pneumoniae subverts the activation of inflammatory responses in a NOD1-dependent manner

Klebsiella pneumoniae is an important cause of community-acquired and nosocomial pneumonia. Subversion of inflammation is essential for pathogen survival during infection. Evidence indicates that K. pneumoniae infections are characterized by lacking an early inflammatory response although the molecular bases are currently unknown. Here we unveil a novel strategy employed by a pathogen to counteract the activation of inflammatory responses. K. pneumoniae attenuates pro-inflammatory mediators-induced IL-8 secretion. Klebsiella antagonizes the activation of NF-κB via the deubiquitinase CYLD and blocks the phosphorylation of mitogen-activated protein kinases (MAPKs) via the MAPK phosphatase MKP-1. Our studies demonstrate that K. pneumoniae has evolved the capacity to manipulate host systems dedicated to control the immune balance. To exert this anti-inflammatory effect, Klebsiella engages NOD1. In NOD1 knock-down cells, Klebsiella neither induces the expression of CYLD and MKP-1 nor blocks the activation of NF-κB and MAPKs. Klebsiella inhibits Rac1 activation; and inhibition of Rac1 activity triggers a NOD1-mediated CYLD and MKP-1 expression which in turn attenuates IL-1β-induced IL-8 secretion. A capsule (CPS) mutant does not attenuate the inflammatory response. However, purified CPS neither reduces IL-1β-induced IL-8 secretion nor induces the expression of CYLD and MKP-1 thereby indicating that CPS is necessary but not sufficient to attenuate inflammation.

Growing Burkholderia pseudomallei in Biofilm Stimulating Conditions Significantly Induces Antimicrobial Resistance

Background Burkholderia pseudomallei, a Gram-negative bacterium that causes melioidosis, was reported to produce biofilm. As the disease causes high relapse rate when compared to other bacterial infections, it therefore might be due to the reactivation of the biofilm forming bacteria which also provided resistance to antimicrobial agents. However, the mechanism on how biofilm can provide tolerance to antimicrobials is still unclear.Methodology/Principal FindingsThe change in resistance of B. pseudomallei to doxycycline, ceftazidime, imipenem, and trimethoprim/sulfamethoxazole during biofilm formation were measured as minimum biofilm elimination concentration (MBEC) in 50 soil and clinical isolates and also in capsule, flagellin, LPS and biofilm mutants. Almost all planktonic isolates were susceptible to all agents studied. In contrast, when they were grown in the condition that induced biofilm formation, they were markedly resistant to all antimicrobial agents even though the amount of biofilm production was not the same. The capsule and O-side chains of LPS mutants had no effect on biofilm formation whereas the flagellin-defective mutant markedly reduced in biofilm production. No alteration of LPS profiles was observed when susceptible form was changed to resistance. The higher amount of N-acyl homoserine lactones (AHLs) was detected in the high biofilm-producing isolates. Interestingly, the biofilm mutant which produced a very low amount of biofilm and was sensitive to antimicrobial agents significantly resisted those agents when grown in biofilm inducing condition.Conclusions/SignificanceThe possible drug resistance mechanism of biofilm mutants and other isolates is not by having biofilm but rather from some factors that up-regulated when biofilm formation genes were stimulated. The understanding of genes related to this situation may lead us to prevent B. pseudomallei biofilms leading to the relapse of melioidosis.

Gamma rays-induced mutant spectrum and frequency in sesame

A comparative study was made to determine the spectrum and frequency of different mutants induced by gamma rays in three genetic backgrounds of sesame and to test the hypothesis that “closed capsule mutants are inducible with efficient mutagenesis and screening large populations”. Treatments concerned seeds of the two cultivars “32-15” and “38-1-7” extensively grown in Senegal and a recently released Turkish cultivar, “Birkan”, irradiated by two doses (300 and 400 Gy) of gamma rays. Both irradiated and untreated seeds (control) were sown greenhouse to raise the M1 populations. The M2 populations produced from the bulked M1 plants at harvest were grown twice under irrigated conditions in two sets; first set in Antalya, Turkey, in 2008 and the second set in Bambey, Senegal, in 2009. The selected potential mutants in the 2-set of M2 populations were grown in the M3 stage in Bambey to confirm their true breeding behaviour. Results indicate that the mutants confirmed shared a wide and unique spectrum of the mutants such as closed capsules, branching habit, flowering types, capsule number and shape. Also at least one closed capsule mutant could be induced from each of the three genetic backgrounds of sesame tested. Among the cultivars tested “38-1-7” had the highest total mutant frequency (5.6 x 10 -3 ) followed by “Birkan” (4.2 x 10 -3 ). The lowest mutant frequency (2.7 x 10 -3 ) was recorded in “32-15”. From these results, it appears that medium dose range of gamma rays

Comparativein vivoandin vitroanalyses of putative virulence factors ofBurkholderia pseudomalleiusing lipopolysaccharide, capsule and flagellin mutants

Burkholderia pseudomallei is a gram-negative bacillus that is the causative agent of melioidosis. We evaluated host-pathogen interaction at different levels using three separate B. pseudomallei mutants generated by insertional inactivation. One of these mutants is defective in the production of the polysaccharide side chains associated with lipopolysaccharide; one does not produce the capsular polysaccharide with the structure -3)-2-O-acetyl-6-deoxy-beta-d-manno-heptopyranose-(1-; and the third mutant does not produce flagellin. We compared the in vivo virulence in BALB/c mice, the in vitro fate of intracellular survival inside human polymorphonuclear cells (PMNs) and macrophages (Mphis) and the susceptibility to killing by 30% normal human serum, reactive nitrogen and oxygen intermediates and antimicrobial peptides with that of their wild-type counterpart. The lipopolysaccharide and capsule mutants demonstrated a marked reduction in virulence for BALB/c mice, but the flagellin mutant was only slightly less virulent than the parent strain. The results from the BALB/c mice experiments correlated with survival in Mphis. The lipopolysaccharide and capsule mutants were also more susceptible to killing by antimicrobial agents. All bacteria were equally susceptible to killing by PMNs. Altogether, the data suggest that lipopolysaccharide and capsule and, to a much lesser extent, flagella, are most likely associated with the virulence of this bacterium and highlight the importance of intracellular killing by PMNs and Mphis in disease pathogenesis.

Vibrio cholerae O139 requires neither capsule nor LPS O side chain to grow inside Acanthamoeba castellanii

Vibrio cholerae, the causative agent of cholera, has the ability to grow and survive in the aquatic free-living amoeba Acanthamoeba castellanii. The aim of the present study was to examine the ability of the clinical isolate V. cholerae O139 MO10 to grow in A. castellanii and to determine the effect of the bacterial capsule and LPS O side chain on intracellular growth. Results from co-cultivation, viable counts, a gentamicin assay, electron microscopy and statistical analysis showed that the association of V. cholerae O139 MO10 with A. castellanii did not inhibit growth of the amoeba, and enhanced growth and survival of V. cholerae O139 MO10 occurred. The wild-type V. cholerae O139 MO10 and a capsule mutant or capsule/LPS double mutant grew inside A. castellanii. Neither the capsule nor the LPS O side chain of V. cholerae O139 was found to play an important role in the interaction with A. castellanii, disclosing the ability of V. cholerae to multiply and survive inside A. castellanii, as well as the role of A. castellanii as an environmental host for V. cholerae.

Determination of Oil Content and Fatty Acid Composition of Sesame Mutants Suited for Intensive Management Conditions

AbstractSesame mutants with closed capsules, determinate growth habit and wilt resistance, have been proposed to be suitable for intensive management conditions facilitating mechanized harvesting. The objective of our experiment was to determine the oil content and fatty acid composition of these mutants before they are placed on the market. Oil content and fatty acids were studied in 19 mutants, 6 breeding lines and 4 control source genotypes. The oil contents of the seeds ranged from 46.4 to 62.7%. The mutants had generally a lower oil content than the control genotypes except the wilting tolerant group. For unsaturated fatty acids, oleic acid was higher in the mutants and breeding lines while linoleic acid was lower in the seed oil. However, no mutants or breeding lines were found with radically different composition or with any undesirable lipid component. The closed capsule and determinate growth habit mutants need to be improved for oil content while their fatty acid composition is fine.

Identification and characterization of the capsular polysaccharide (K-antigen) locus of Porphyromonas gingivalis.

Capsular polysaccharides of gram-negative bacteria play an important role in maintaining the structural integrity of the cell in hostile environments and, because of their diversity within a given species, can act as useful taxonomic aids. In order to characterize the genetic locus for capsule biosynthesis in the oral gram-negative bacterium Porphyromonas gingivalis, we analyzed the genome of P. gingivalis W83 which revealed two candidate loci at PG0106-PG0120 and PG1135-PG1142 with sufficient coding capacity and appropriate gene functions based on comparisons with capsule-coding loci in other bacteria. Insertion and deletion mutants were prepared at PG0106-PG0120 in P. gingivalis W50-a K1 serotype. Deletion of PG0109-PG0118 and PG0116-PG0120 both yielded mutants which no longer reacted with antisera to K1 serotypes. Restriction fragment length polymorphism analysis of the locus in strains representing all six K-antigen serotypes and K(-) strains demonstrated significant variation between serotypes and limited conservation within serotypes. In contrast, PG1135-PG1142 was highly conserved in this collection of strains. Sequence analysis of the capsule locus in strain 381 (K(-) strain) demonstrated synteny with the W83 locus but also significant differences including replacement of PG0109-PG0110 with three unique open reading frames, deletion of PG0112-PG0114, and an internal termination codon within PG0106, each of which could contribute to the absence of capsule expression in this strain. Analysis of the Arg-gingipains in the capsule mutants of strain W50 revealed no significant changes to the glycan modifications of these enzymes, which indicates that the glycosylation apparatus in P. gingivalis is independent of the capsule biosynthetic machinery.

Characterizing lipopolysaccharide and core lipid A mutant O1 and O139 Vibrio cholerae strains for adherence properties on mucus-producing cell line HT29-Rev MTX and virulence in mice

Components of lipopolysaccharide (LPS), i.e. capsule, O antigen, core oligosaccharide, as well as the toxin-coregulated pili are among the factors which significantly contribute to intestinal colonization by Vibrio cholerae O1 and O139. To further address the contribution of LPS to V. cholerae virulence, we performed in vivo colonization experiments and mucus layer attachment studies with defined LPS and capsule mutants of O1 and O139. We investigated the interaction of V. cholerae strains with the differentiated human intestinal cell line HT29-Rev MTX, and found 3–5-fold reduced efficiencies for attachment by defined LPS and capsule mutants of O1 and O139 in comparison with the wild-type strains. In addition, two O1/O139-specific core oligosaccharide biosynthetic gene products, WavJ and WavD, were characterized and tested for colonization. We demonstrate that single and double knockout mutants in wavJ and wavD have an effect on core oligosaccharide biosynthesis, and that these mutants show an attenuated growth in the presence of novobiocin. Curiously, in the mouse intestinal colonization model, only the O139 wavJ,D mutants demonstrated reduced colonization.

Large-Scale Screen Highlights the Importance of Capsule for Virulence in the Zoonotic PathogenStreptococcus iniae

Zoonotic pathogens have the unique ability to cross the species barrier, causing disease in both humans and specific animal hosts. Streptococcus iniae is a zoonotic pathogen of both fish and humans, and the clinical presentations of S. iniae infections in fish and humans are very similar to those caused by various human-specific streptococcal pathogens. Virulence mechanisms required for infection by this pathogen of either host have yet to be determined. Using the previously reported zebrafish infectious disease model, we performed a large-scale screening to determine genes required for systemic infection. Screening 1,128 signature-tagged transposon mutants through the zebrafish model allowed identification of 41 potential mutants that were unable to survive within the host environment. Greater than 50% of the mutants that could be identified through homology searches were highly homologous to genes found in other human-specific streptococcal pathogens, while 32% were found to have no homology to any sequences found in the databases, suggesting as yet unknown gram-positive bacterial virulence factors. A large percentage of the insertions were found to be located in several putative capsule synthesis genes, an important virulence component for other systemic pathogens. Density gradient assays demonstrated that several of these putative capsule mutants have dissimilar buoyant densities, suggesting different levels of capsule synthesis. Putative capsule mutants were also less resistant to phagocytosis in whole-blood assays than wild-type S. iniae. Our initial large-scale characterization of S. iniae virulence highlights the importance of the capsule for successful infection.

The Capsular Polysaccharide ofBurkholderia pseudomalleiContributes to Survival in Serum by Reducing Complement Factor C3b Deposition

Burkholderia pseudomallei produces an extracellular polysaccharide capsule -3)-2-O-acetyl-6-deoxy-beta-D-manno-heptopyranose-(1- which has been shown to be an essential virulence determinant. The addition of purified capsule was shown to increase the virulence of a capsule mutant strain in the Syrian hamster model of acute melioidosis. An increase in the number of wild-type B. pseudomallei cells in the blood was seen by 48 h, while the number of capsule mutant cells in the blood declined by 48 h. Capsule expression was shown to be induced in the presence of serum using a lux reporter fusion to the capsule gene wcbB. The addition of purified B. pseudomallei capsule to serum bactericidal assays increased the survival of B. pseudomallei SLR5, a serum-sensitive strain, by 1,000-fold in normal human serum. Capsule production by B. pseudomallei contributed to reduced activation of the complement cascade by reducing the levels of complement factor C3b deposition. An increase in phagocytosis of the capsule mutant compared to the wild type was observed in the presence of normal human serum. These results suggest that the production of this capsule contributes to resistance to phagocytosis by reducing C3b deposition on the surface of the bacterium, thereby contributing to the persistence of bacteria in the blood of the infected host. Continued studies to characterize this capsule are essential for understanding the pathogenesis of B. pseudomallei infections and the development of preventive strategies for treatment of this disease.

Capsule synthesis by Bacillus anthracis is required for dissemination in murine inhalation anthrax

Bacillus anthracis, the agent of anthrax, produces a poly-D-glutamic acid capsule that has been implicated in virulence. Many strains missing pXO2 (96 kb), which harbors the capsule biosynthetic operon capBCAD, but carrying pXO1 (182 kb) that harbors the anthrax toxin genes, are attenuated in animal models. Also, noncapsulated strains are readily phagocytosed by macrophage cell lines, whereas capsulated strains are resistant to phagocytosis. We show that a strain carrying both virulence plasmids but deleted specifically for capBCAD is highly attenuated in a mouse model for inhalation anthrax. The parent strain and capsule mutant initiated germination in the lungs, but the capsule mutant did not disseminate to the spleen. A mutant harboring capBCAD but deleted for the cap regulators acpA and acpB was also significantly attenuated, in agreement with the capsule-negative phenotype during in vitro growth. Surprisingly, an acpB mutant, but not an acpA mutant, displayed an elevated LD(50) and reduced ability to disseminate, indicating that acpA and acpB are not true functional homologs and that acpB may play a larger role in virulence than originally suspected.

Aerogenic vaccination with a Burkholderia mallei auxotroph protects against aerosol-initiated glanders in mice

Burkholderia mallei is an obligate mammalian pathogen that causes the zoonotic disease glanders. Two live attenuated B. mallei strains, a capsule mutant and a branched-chain amino acid auxotroph, were evaluated for use as vaccines against aerosol-initiated glanders in mice. Animals were aerogenically vaccinated and serum samples were obtained before aerosol challenge with a high-dose (>300 times the LD 50) of B. mallei ATCC 23344. Mice vaccinated with the capsule mutant developed a Th2-like Ig subclass antibody response and none survived beyond 5 days. In comparison, the auxotrophic mutant elicited a Th1-like Ig subclass antibody response and 25% of the animals survived for 1 month postchallenge. After a low-dose (5 times the LD 50) aerosol challenge, the survival rates of auxotroph-vaccinated and unvaccinated animals were 50 and 0%, respectively. Thus, live attenuated strains that promote a Th1-like Ig response may serve as promising vaccine candidates against aerosol infection with B. mallei.