Capsule Gene Expression Research Articles

Leveraging a new data resource to define the response of C. neoformans to environmental signals.

Cryptococcus neoformans is an opportunistic fungal pathogen with a polysaccharide capsule that becomes greatly enlarged in the mammalian host and during in vitro growth under host-like conditions. To understand how individual environmental signals affect capsule size and gene expression, we grew cells in all combinations of five signals implicated in capsule size and systematically measured cell and capsule sizes. We also sampled these cultures over time and performed RNA-Seq in quadruplicate, yielding 881 RNA-Seq samples. Analysis of the resulting data sets showed that capsule induction in tissue culture medium, typically used to represent host-like conditions, requires the presence of either CO2 or exogenous cyclic AMP (cAMP). Surprisingly, adding either of these pushes overall gene expression in the opposite direction from tissue culture media alone, even though both are required for capsule development. Another unexpected finding was that rich medium blocks capsule growth completely. Statistical analysis further revealed many genes whose expression is associated with capsule thickness; deletion of one of these significantly reduced capsule size. Beyond illuminating capsule induction, our massive, uniformly collected dataset will be a significant resource for the research community.

Shedding light on Klebsiella pneumoniae virulence: Engineering of broad host range bioluminescence reporter vectors for transcriptional analysis in drug resistant pathogens.

In this work, we report the construction of four bacterial luciferase-based promoter probe vectors with an expanded set of selectable markers, designed to facilitate their use in antibiotic-resistant bacteria. These vectors contain the low-copy-number, broad-host-range pBBR origin of replication and an origin of transfer, allowing efficient conjugative transformation into various bacterial genera. The broad host range origin also enables their use in bacterial strains that harbor other plasmids, as the pBBR origin is compatible with a wide variety of other plasmid replication systems. The utility of these vectors was demonstrated by quantifying capsule gene expression in both classical and hypervirulent Klebsiella pneumoniae strains lacking tolC, which encodes the outer membrane pore protein for tripartite transport systems. Our results revealed that the tolC mutation reduced capsule gene expression, highlighting a critical role for tolC in K. pneumoniae pathobiology and the utility of bioluminescence for studying gene expression in real time. These new vectors provide a flexible platform for circumventing antibiotic resistance phenotypes and studying gene expression across diverse bacterial species, including strains containing additional plasmids.

YbdO Promotes the Pathogenicity of Escherichia coli K1 by Regulating Capsule Synthesis.

Escherichia coli K1 is the most popular neonatal meningitis-causing Gram-negative bacterium. As a key virulence determinant, the K1 capsule enhances the survival of E. coli K1 in human brain microvascular endothelial cells (HBMECs) upon crossing the blood–brain barrier; however, the regulatory mechanisms of capsule synthesis during E. coli K1 invasion of HBMECs remain unclear. Here, we identified YbdO as a transcriptional regulator that promotes E. coli K1 invasion of HBMECs by directly activating K1 capsule gene expression to increase K1 capsule synthesis. We found that ybdO deletion significantly reduced HBMEC invasion by E. coli K1 and meningitis occurrence in mice. Additionally, electrophoretic mobility shift assay and chromatin immunoprecipitation–quantitative polymerase chain reaction analysis indicated that YbdO directly activates kpsMT and neuDBACES expression, which encode products involved in K1 capsule transport and synthesis by directly binding to the kpsM promoter. Furthermore, ybdO transcription was directly repressed by histone-like nucleoid structuring protein (H-NS), and we observed that acidic pH similar to that of early and late endosomes relieves this transcriptional repression. These findings demonstrated the regulatory mechanism of YbdO on K1 capsule synthesis, providing further insights into the evolution of E. coli K1 pathogenesis and host–pathogen interaction.

Regulation of Escherichia coli Group 2 Capsule Gene Expression: A Mini Review and Update.

The expression of a group 2 capsule (K antigen), such as the K1 or K5 antigen, is a key virulence factor of Escherichia coli responsible for extra-intestinal infections. Capsule expression confers resistance to innate host defenses and plays a critical role in invasive disease. Capsule expression is temperature-dependent being expressed at 37°C but not at 20°C when outside the host. Group 2 capsule gene expression involves two convergent promoters PR1 and PR3, the regulation of which is critical to capsule expression. Temperature-dependent expression is controlled at transcriptional level directly by the binding of H-NS to PR1 and PR3 and indirectly through BipA with additional input from IHF and SlyA. More recently, other regulatory proteins, FNR, Fur, IHF, MprA, and LrhA, have been implicated in regulating capsule gene expression in response to other environmental stimuli and there is merging data for the growth phase-dependent regulation of the PR1 and PR3 promoters. The aim of the present Mini Review is to provide a unified update on the latest data on how the expression of group 2 capsules is regulated in response to a number of stimuli and the growth phase something that has not to date been addressed.

Whole genome and RNA sequencing of oral commensal bacterium Streptococcus anginosus subsp. anginosus with vancomycin tolerance.

"Antibiotic tolerance" promotes the rapid subsequent evolution of "antibiotic resistance," however, it is often overlooked because it is difficult to distinguish between tolerant and susceptible organisms. A commensal bacterium S. anginosus subsp. anginosus strain KHUD_S1, isolated from dental biofilm was found to exhibit a high MBC/MIC ratio of 32 against vancomycin. We observed KHUD_S1 cells exposed to vancomycin did not grow but maintained viability. Transmission electron microscope showed KHUD_S1 cells possessed a dense, thick capsule and maintained the cell wall integrity upon vancomycin exposure. To infer the underlying mechanisms of the vancomycin tolerance in KHUD_S1, we performed whole genome sequencing and RNA sequencing. The KHUD_S1 genome carried three genes encoding branching enzymes that can affect peptidoglycan structure through interpeptide bridge formation. Global gene expression profiling revealed that the vancomycin-induced downregulation of carbohydrate and inorganic ion transport/metabolism as well as translation is less prominent in KHUD_S1 than in the vancomycin susceptible strain KHUD_S3. Based on the transcriptional levels of genes related to peptidoglycan synthesis, KHUD_S1 was determined to have a 3D peptidoglycan architecture distinct from KHUD_S3. It was found that, under vancomycin exposure, the peptidoglycan was remodeled through changes in the interpeptide bridge and transpeptidation reactions. Collectively, these features of S. anginosus KHUD_S1, including a dense capsule and differential gene expression in peptidoglycan synthesis, may contribute to vancomycin tolerance. Our results showing the occurrence of vancomycin tolerance amongst oral commensal bacteria highlight the need for considering future strategies for screening of antibiotic tolerance as an effort to reduce antibiotic resistance.

The Small Protein RmpD Drives Hypermucoviscosity in Klebsiella pneumoniae.

Klebsiella pneumoniae has a remarkable ability to cause a wide range of human diseases. It is divided into two broad classes: classical strains that are a notable problem in health care settings due to multidrug resistance, and hypervirulent (hv) strains that are historically drug sensitive but able to establish disease in immunocompetent hosts. Alarmingly, there has been an increased frequency of clinical isolates that have both drug resistance and hv-associated genes. One such gene, rmpA, encodes a transcriptional regulator required for maximal capsule (cps) gene expression and confers hypermucoviscosity (HMV). This link has resulted in the assumption that HMV is caused by elevated capsule production. However, we recently reported a new cps regulator, RmpC, and ΔrmpC mutants have reduced cps expression but retain HMV, suggesting that capsule production and HMV may be separable traits. Here, we report the identification of a small protein, RmpD, that is essential for HMV but does not impact capsule. RmpD is 58 residues with a putative N-terminal transmembrane domain and highly positively charged C-terminal half, and it is conserved among other hv K. pneumoniae strains. Expression of rmpD in trans complements both ΔrmpD and ΔrmpA mutants for HMV, suggesting that RmpD is the key driver of this phenotype. The rmpD gene is located between rmpA and rmpC, within an operon regulated by RmpA. These data, combined with our previous work, suggest a model in which the RmpA-associated phenotypes are largely due to RmpA activating the expression of rmpD to produce HMV and rmpC to stimulate cps expression.IMPORTANCE Capsule is a critical virulence factor in Klebsiella pneumoniae, in both antibiotic-resistant classical strains and hypervirulent strains. Hypervirulent strains usually have a hypermucoviscosity (HMV) phenotype that contributes to their heightened virulence capacity, but the production of HMV is not understood. The transcriptional regulator RmpA is required for HMV and also activates capsule gene expression, leading to the assumption that HMV is caused by hyperproduction of capsule. We have identified a new gene (rmpD) required for HMV but not for capsule production. This distinction between HMV and capsule production will promote a better understanding of the mechanisms of hypervirulence, which is in great need given the alarming increase in clinical isolates with both drug resistance and hypervirulence traits.

Carbon source regulates polysaccharide capsule biosynthesis in Streptococcus pneumoniae

The exopolysaccharide capsule of Streptococcus pneumoniae is an important virulence factor, but the mechanisms that regulate capsule thickness are not fully understood. Here, we investigated the effects of various exogenously supplied carbohydrates on capsule production and gene expression in several pneumococcal serotypes. Microscopy analyses indicated a near absence of the capsular polysaccharide (CPS) when S. pneumoniae was grown on fructose. Moreover, serotype 7F pneumococci produced much less CPS than strains of other serotypes (6B, 6C, 9V, 15, and 23F) when grown on glucose or sucrose. RNA-sequencing revealed carbon source-dependent regulation of distinct genes of WT strains and capsule-switch mutants of serotypes 6B and 7F, but could not explain the mechanism of capsule thickness regulation. In contrast, 31P NMR of whole-cell extract from capsule-knockout strains (Δcps) clearly revealed the accumulation or absence of capsule precursor metabolites when cells were grown on glucose or fructose, respectively. This finding suggests that fructose uptake mainly results in intracellular fructose 1-phosphate, which is not converted to CPS precursors. In addition, serotype 7F strains accumulated more precursors than did 6B strains, indicating less efficient conversion of precursor metabolites into the CPS in 7F, in line with its thinner capsule. Finally, isotopologue sucrose labeling and NMR analyses revealed that the uptake of the labeled fructose subunit into the capsule is <10% that of glucose. Our findings on the effects of carbon sources on CPS production in different S. pneumoniae serotypes may contribute to a better understanding of pneumococcal diseases and could inform future therapeutic approaches.

Identification of Two Regulators of Virulence That Are Conserved in Klebsiella pneumoniae Classical and Hypervirulent Strains.

ABSTRACTKlebsiella pneumoniae is widely recognized as a pathogen with a propensity for acquiring antibiotic resistance. It is capable of causing a range of hospital-acquired infections (urinary tract infections [UTI], pneumonia, sepsis) and community-acquired invasive infections. The genetic heterogeneity of K. pneumoniae isolates complicates our ability to understand the virulence of K. pneumoniae. Characterization of virulence factors conserved between strains as well as strain-specific factors will improve our understanding of this important pathogen. The MarR family of regulatory proteins is widely distributed in bacteria and regulates cellular processes such as antibiotic resistance and the expression of virulence factors. Klebsiella encodes numerous MarR-like proteins, and they likely contribute to the ability of K. pneumoniae to respond to and survive under a wide variety of environmental conditions, including those present in the human body. We tested loss-of-function mutations in all the marR homologues in a murine pneumonia model and found that two (kvrA and kvrB) significantly impacted the virulence of K1 and K2 capsule type hypervirulent (hv) strains and that kvrA affected the virulence of a sequence type 258 (ST258) classical strain. In the hv strains, kvrA and kvrB mutants displayed phenotypes associated with reduced capsule production, mucoviscosity, and transcription from galF and manC promoters that drive expression of capsule synthesis genes. In contrast, kvrA and kvrB mutants in the ST258 strain had no effect on capsule gene expression or capsule-related phenotypes. Thus, KvrA and KvrB affect virulence in classical and hv strains but the effect on virulence may not be exclusively due to effects on capsule production.

Generating a SlyA Mutation in E. Coli and Validating Its Effect on Microbial Response to Environmental Stress Factors

Background: In the human host, environments encountered by bacteria can vary widely. Bacteria experience stress from their initial moment of contact with the host. Therefore, adaptation and survival of the bacteria hinge on their ability to probe the environment and respond appropriately. Capsule expression is one of the responses to stress. In E. coli K5 capsule expression is temperature regulated and SlyA was proven to be involved in E. coli capsule gene expression. Methodology: Using HA1 cells as a model, cells were grown under different environmental conditions using a set of chemical substances and the b-galactosidase activity was measured and used as an indicator of capsule expression. pGEM-T easy vector and a series of PCR were used in cloning and generating a tet-insertional mutation of the slyA gene. Results: Maximum activity of capsule expression was under anaerobic conditions, then microaerophilic with least activity been obtained aerobically. Maximum capsule expression was obtained in presence of 0.4M sucrose or NaCl and 2mM MgSO 4 at pH 7.5. In all conditions capsule expression was increased ~2.4 fold in the presence of multicopy slyA plasmid; an effect that disappeared with tet:: slyA. Conclusion: Intestinal environmental cues in terms of anaerobiosis, relatively high osmolarity in addition to the slightly alkaline pH, all can contribute to up-regulating capsule gene expression in E. coli intestinal flora. Many aspects of clinical host-pathogen interactions involve stress responses and adaptive responses allow different pathogenic bacteria to resist host defences. Capsule expression was significantly increased in the presence of a multicopy slyA

Phenotypic Heterogeneity in Expression of the K1 Polysaccharide Capsule of Uropathogenic Escherichia coli and Downregulation of the Capsule Genes during Growth in Urine.

Uropathogenic Escherichia coli (UPEC) is the major causative agent of uncomplicated urinary tract infections (UTI). The K1 capsule on the surface of UPEC strains is a key virulence factor, and its expression may be important in the onset and progression of UTI. In order to understand capsule expression in more detail, we analyzed its expression in the UPEC strain UTI89 during growth in rich medium (LB medium) and urine and during infection of a bladder epithelial cell line. Comparison of capsule gene transcription using a chromosomal gfp reporter fusion showed a significant reduction in transcription during growth in urine compared to that during growth in LB medium. When examined at the single-cell level, following growth in both media, capsule gene expression appears to be heterogeneous, with two distinct green fluorescent protein (GFP)-expressing populations. Using anti-K1 antibody, we showed that this heterogeneity in gene expression results in two populations of encapsulated and unencapsulated cells. We demonstrated that the capsule hinders attachment to and invasion of epithelial cells and that the unencapsulated cells within the population preferentially adhere to and invade bladder epithelial cells. We found that once internalized, UTI89 starts to produce capsule to aid in its intracellular survival and spread. We propose that this observed phenotypic diversity in capsule expression is a fitness strategy used by the bacterium to deal with the constantly changing environment of the urinary tract.

Bis-(3′-5′)-cyclic dimeric GMP-linked quorum sensing controls swarming in Vibrio parahaemolyticus

Movement over and colonization of surfaces are important survival strategies for bacteria, and many find it advantageous to perform these activities as a group, using quorum sensing to sample population size and synchronize behavior. It is puzzling however, that swarming-proficient and virulent strains of Vibrio parahaemolyticus are silenced for the vibrio archetypal pathway of quorum sensing. Here we describe the S-signal, a pheromone that can be communicated between cells in coculture to regulate surface colonization. This signal was harvested in cell-free supernatants and demonstrated to stimulate swarming gene expression at low cell density. The S-signal was generated by the pyridoxal phosphate-dependent aminotransferase ScrA; signal reception required the periplasmic binding protein ScrB and the membrane-bound GGDEF-EAL domain-containing protein ScrC. ScrC is a bifunctional enzyme that has the ability to form and degrade the second messenger bis-(3'-5') cyclic dimeric GMP (c-di-GMP). ScrA in neighboring cells was able to alter the activity of ScrC in a ScrB-dependent manner, transforming ScrC's repressing ability to inducing activity with respect to swarming. Conversely, cell-cell signaling repressed capsule gene expression. In summary, we report that quorum sensing can stimulate swarming in V. parahaemolyticus; it does so via an alternative pathway capable of generating an autoinducing signal that influences c-di-GMP, thereby expanding the lexicon and language of cell-cell communication.

Characterization of the type III capsular polysaccharide produced by Burkholderia pseudomallei

Burkholderia pseudomallei has been shown to produce more than one capsular polysaccharide (CPS). Analysis of the B. pseudomallei genome has revealed that the organism contains four CPS operons (I-IV). One of these operons (CPS III) was selected for further study. Comparative sequencing analysis revealed that the genes encoding CPS III are present in B. pseudomallei and Burkholderia thailandensis but not in Burkholderia mallei. In this study, CPS III was not found to contribute to the virulence of B. pseudomallei. Strains containing mutations in CPS III had the same LD(50) value as the wild-type when tested in an animal infection model. Production of CPS III was shown to be induced in water but inhibited in 30% normal human serum using a lux reporter fusion assay. Microarray analysis of capsule gene expression in infected hamsters revealed that the genes encoding CPS III were not significantly expressed in vivo compared with the genes encoding the previously characterized mannoheptose capsule (CPS I), which is an important virulence factor in B. pseudomallei. Glycosyl-composition analysis by combined GC/MS indicated that the CPS III genes are involved in the synthesis of a capsule composed of galactose, glucose, mannose and xylose.

Xylitol and capsular gene expression in Streptococcus pneumoniae

Xylitol is a sugar alcohol that inhibits the growth and adherence of Streptococcus pneumoniae. In clinical trials, xylitol has been shown to decrease the occurrence of acute otitis media in day-care children but did not decrease nasopharyngeal carriage of the pneumococci. It has also been shown that xylitol affects the ultrastructure of the pneumococcal capsule. Here, it was hypothesized that xylitol might affect the expression of pneumococcal capsular genes. Capsule gene expression levels were studied in 24 clinical pneumococcal isolates and one ATCC strain (49619) by using a real-time RT-PCR method targeting the mRNA of the second gene of the pneumococcal capsular locus, the cpsB gene. The isolates were exposed to 5 % glucose, 5 % xylitol and control medium (brain heart infusion medium containing 10 % fetal bovine serum) for 2 h. cpsB gene expression levels were measured by using a relative quantification method with calibrator normalization where the 16S rRNA gene of pneumococcus was used as a reference. Exposure to xylitol lowered cpsB gene expression levels significantly compared with those in the control (P=0.035) and glucose (P=0.011) media. This finding supports previous results where exposure to xylitol changed the ultrastructure of the pneumococcal capsule and could explain further the high clinical efficacy of xylitol in preventing otitis media.

Dual Promoters Control Expression of theBacillus anthracisVirulence Factor AtxA

The AtxA virulence regulator of Bacillus anthracis is required for toxin and capsule gene expression. AtxA is a phosphotransferase system regulatory domain-containing protein whose activity is regulated by phosphorylation/dephosphorylation of conserved histidine residues. Here we report that transcription of the atxA gene occurs from two independent promoters, P1 (previously described by Dai et al. [Z. Dai, J. C. Sirard, M. Mock, and T. M. Koehler, Mol. Microbiol. 16:1171-1181, 1995]) and P2, whose transcription start sites are separated by 650 bp. Both promoters have -10 and -35 consensus sequences compatible with recognition by sigma(A)-containing RNA polymerase, and neither promoter depends on the sporulation sigma factor SigH. The dual promoter activity and the extended untranslated mRNA suggest that as-yet-unknown regulatory mechanisms may act on this region to influence the level of AtxA in the cell.

209 INFLUENCE OF OP-1 and IGF-1 ON CARTILAGE SUBJECTED TO COMBINED MECHANICAL INJURY AND CO-CULTURE WITH JOINT CAPSULE

Introduction: Progression to osteoarthritis after joint injury involves multiple tissue interactions. The combination of cartilage mechanical injury (INJ) in vitro followed by co-culture with excised joint capsule (Co) has been used as a model to physiologically mimic an acute injury [1]. Previous studies [2] have shown that INJ+Co increases catabolic gene expression and aggrecan fragment levels. We hypothesized that treatment of growth factors will mitigate the effects of injury on gene, protein, and cell vitality levels. Our objective was to test whether OP-1 and IGF-1 can protect cartilage and joint capsule ( JC) following injury by measuring gene expression, proteoglycan synthesis, and apoptosis. Materials and Methods: Tissue Harvest: Bovine calf cartilage disks were harvested from the patello-femoral groove and equilibrated for 2 days under free-swell (FS) conditions in serum-free DMEM supplemented with 1% ITS. JC explants were excised adjacent to the femoral condyles and equilibrated for two days. Injury and Time Course: At time = 0, cartilage disks were allocated into one of 8 conditions; (1) FS, (2) normal cartilage co-cultured with JC (Co), (3) cartilage mechanically injured (INJ, 50% strain at 1mm/sec), and (4) injured cartilage co-cultured with joint capsule (INJ+Co), (5-8) 100ng/ml OP-1 + 300ng/ml IGF-1 (GFs) added throughout culture duration. Gene Expression: Cartilage disks and JC were flash frozen 2, 8, 24, 48, and 72 hours after injury for short term, and frozen at days 1, 4,8, and 16 days for longer term; RNA was extracted, reverse-transcribed, and mRNA levels of 48 cartilage relevant genes measured using qPCR and normalized to 18s. K-means-Clustering analysis was performed to determine gene co-expression patterns. Protein Biosynthesis: 35S-sulfate and 3H-proline incorporation (for proteoglycans and total protein synthesis) were assessed in the last 24 hours of 1, 4, 8, 12, or 16 day cultures post injury. Apoptosis: Plugs were fixed in 4% paraformaldahyde at 1, 4, 8, and 16 days post injury and were sectioned to evaluate apoptosis via nuclear blebbing by light microscopy. Statistics: Significance was determined using the Wilcoxon-Sign ranked Test and ANOVA at the p < 0.05 level. Results: Cartilage Short Term Gene Expression: Key ECM molecules (aggrecan, collagens) were significantly down-regulated in the presence of Co, INJ, and INJ+Co. Proteinases (MMP-1,3,9,13; ADAMTS-1,4,5) and TIMP-1,2,3 were maximally expressed compared to controls by INJ+Co. GF failed to rescue ECM down-regulation, while up-regulating proteinase transcripts. iNOS and caspace-3 were maximally expressed under Co alone; iNOS reached values of 2000x FS. Clustering analysis revealed 5 distinct gene expression profiles (Fig 1): Group-1 (ECM molecules) was down-regulated with Co, INJ, and INJ+Co. Group-2 (iNOS, Cas-3) was up-regulated by Co. Groups-3, 5 (proteases) were maximally expressed by INJ+Co. Group-4 (oxidation genes) was responsive to GF treatment. Joint Capsule Gene Expression: Relative gene expression levels within JC explants while in co-culture with normal cartilage explants showed proportionally higher levels of protease abundance compared to normal cartilage (Fig 2). All genes in JC (except ADAMTS-5) were stimulated by GF treatment. Cartilage Longer Term Gene Expression: While most genes measured (Fig 2B) returned to FS values after 16 days, MMP-9 showed a consistent upregulation even at day 16 for Co (147x) and INJ+Co (45x). Chondrocyte Biosynthesis: 35S-sulfate and 3H-proline incorporation was significantly reduced by Co and INJ+Co. GF treatment slightly increased 35S-sulfate incorporation for all conditions (Figure 3A). Apoptosis: Significant apoptosis was observed at days 4, 8, and 16. At Day 8, INJ caused significantly higher levels of apoptosis (47%) compared with FS(<1%). GF treatment significantly decreased apoptosis in INJ alone (Fig 3B). Discussion: GF treatment stimulated most of the genes measured, suggesting that the growth factor combination upregulated general remodeling rather than just anabolic stimulation of ECM. Aggrecan and collagen transcript levels were severely down regulated under each injury stimuli, and GF treatment failed to protect such decreases in transcript levels. GF was able to increase protein synthesis (ongoing studies using IHC are to identify specific anabolic and catabolic events). The joint capsule expressed proteases at levels orders of magnitude greater than non-injured cartilage (Fig 2), suggesting the probability of cartilage-JC communication. GF down-regulated ADAMTS-5 while up-regulating all other genes; suggesting a potentially important protective effect. Caspase-3, a key signaling molecule in apoptosis, was significantly up-regulated (12x) under Co alone. As seen before [3], apoptosis was significantly increased following INJ, while there is a trend towards an increase in cell death under Co. While previous studies showed increased apoptosis due to mechanical injury, we have now shown a direct link between chondrocyte apoptosis in normal cartilage due to co-culture with excised joint capsule. GF treatment protected cartilage against apoptosis under INJ alone; when JC was present the effect was less dramatic. Thus, GFs could party reverse injury-induced changes in biosynthesis and apoptosis, but not transcript levels.

The arl locus positively regulates Staphylococcus aureus type 5 capsule via an mgrA-dependent pathway

Most clinical Staphylococcus aureus strains produce either type 5 or type 8 capsular polysaccharides. The production of these capsules is influenced by various environmental factors. To study the regulation of capsule, Tn551 transposon mutagenesis and transcriptional reporter gene fusion were employed to identify several putative regulatory loci that influenced capsule gene expression. One of these, the arl locus, was chosen for further analysis. Tn551 was found to insert within the coding region (near the translational start site of the arlR gene). ArlR, along with ArlS, forms a two-component system that has been previously shown to affect autolysis and production of several secreted proteins. Phenotypic analyses of the arlR-specific mutant and gene fusion analyses showed that arlR activated capsule production at the transcriptional level. However, gel mobility shift assays did not support activation of the capsule genes by direct ArlR binding to the primary cap5 promoter region upstream of the operon. In contrast, it was found that arl activated mgrA, an activator for capsule production, whereas mgrA did not have a significant effect on arlR. Genetic studies supported the notion that arlR functions upstream of mgrA with respect to the regulation of capsule production, although gene fusion studies indicated that arl could also regulate capsule independently from mgrA. Collectively, the results suggest that arl positively regulates capsule production at the transcriptional level primarily through an mgrA-dependent pathway.

Genetic Analysis of Meningococci Carried by Children and Young Adults

Neisseria meningitidis is a diverse commensal bacterium that occasionally causes severe invasive disease. The relationship between meningococcal genotype and capsular polysaccharide, the principal virulence factor and vaccine component, was investigated in carried meningococci isolated from 8000 children and young adults in Bavaria, Germany. Of the 830 meningococci isolated (carriage rate, 10.4%) by microbiological techniques, 822 were characterized by serogrouping, multilocus sequence typing, and genetic analysis of the capsule region. Statistical and population genetic analyses were applied to these data. The rapid increase in carriage rates with age of carrier, the low prevalence of hyperinvasive meningococci, and the relative prevalence of the 4 disease-associated serogroups were consistent with earlier observations. There was no genetic structuring of the meningococcal population by age of carrier or sampling location; however, there was significant geographic structuring of the meningococci isolated in civil, but not military, institutions. The rate of capsule gene expression did not vary with age of carrier or meningococcal genotype, except for serogroup C, for which increased expression was associated with ST-11 (formerly ET-37) complex meningococci. Serogroup C capsule expression during carriage may contribute to the invasive character of ST-11 complex meningococci and to the high efficacy of meningococcal serogroup C conjugate polysaccharide vaccine.

AtxA controls Bacillus anthracis capsule synthesis via acpA and a newly discovered regulator, acpB.

Two regulatory genes, acpA and atxA, have been reported to control expression of the Bacillus anthracis capsule biosynthesis operon capBCAD. The atxA gene is located on the virulence plasmid pXO1, while pXO2 carries acpA and the cap genes. acpA has been viewed as the major regulator of the cap operon because it is essential for capsule gene expression in a pXO1(-) pXO2(+) strain. atxA is essential for toxin gene transcription but has also been implicated in control of the cap genes. The molecular functions of the regulatory proteins are unknown. We examined cap gene expression in a genetically complete pXO1(+) pXO2(+) strain. Our results indicate that another pXO2 gene, acpB (previously called pXO2-53; accession no. NC002146.1:49418-50866), has a role in cap expression. The predicted amino acid sequence of AcpB is 62% similar to that of AcpA and 50% similar to that of AtxA. Assessment of cap gene transcription revealed that cap expression was not affected in a pXO1(+) pXO2(+) acpB-null mutant and was slightly reduced in an isogenic acpA mutant. However, cap gene expression was abolished in an acpA acpB double mutant. Microscopic examination of capsule synthesis by the mutants corroborated these findings. acpA and acpB expression is controlled by atxA; capsule synthesis and transcription of acpA and acpB were markedly reduced in an atxA mutant. The data suggest that, in a strain containing both virulence plasmids, atxA is the major regulator of capsule synthesis and controls capBCAD expression indirectly, via positive regulation of acpA and acpB.

Analysis of RogB-controlled virulence mechanisms and gene repression in Streptococcus agalactiae.

Streptococcus agalactiae is the leading cause of bacterial sepsis and meningitis in neonates and also the causative agent of different serious infections in immunocompromised adults. The wide range of diseases that are caused by S. agalactiae suggests regulatory mechanisms that control the formation of specific virulence factors in these bacteria. The present study describes a gene from S. agalactiae, designated rogB, encoding a protein with significant similarity to members of the RofA-like protein (RALP) family of transcriptional regulators. Disruption of the rogB gene in the genome of S. agalactiae resulted in mutant strain RGB1, which was impaired in its ability to bind to fibrinogen and fibronectin. Mutant RGB1 also exhibited a reduced adherence to human epithelial cells but did not show an altered invasion of eukaryotic cells. By real-time PCR analysis, mutant RGB1 revealed an increased expression of the cpsA gene, encoding a regulator of capsule gene expression. However, strain RGB1 exhibited a reduced expression of the rogB gene and of two adjacent genes, encoding putative virulence factors in S. agalactiae. Furthermore, mutant RGB1 was impaired in the expression of the fbsA gene, coding for a fibrinogen receptor from S. agalactiae. The altered gene expression in mutant RGB1 could be restored by plasmid-mediated expression of rogB, confirming a RogB deficiency as the cause for the observed changes in virulence gene expression in S. agalactiae. Reporter gene studies with a promotorless luciferase gene fused to fbsA allowed a growth-dependent analysis of fbsA expression in S. agalactiae. These reporter gene studies also suggest that RogB exerts a positive effect on fbsA expression in S. agalactiae.

The CsrR/CsrS two-component system of group A Streptococcus responds to environmental Mg2+.

Group A streptococci control expression of key virulence determinants via the two-component sensorregulator system CsrRCsrS. The membrane-bound sensor CsrS is thought to respond to previously unknown environmental signal(s) by controlling phosphorylation of its cognate regulator component CsrR. Phosphorylation of CsrR increases its affinity for binding to the promoter regions of Csr-regulated genes to repress transcription. Here we show that environmental Mg(2+) concentration is a potent and specific stimulus for CsrRCsrS-mediated regulation. We studied the effect of divalent cations on expression of the Csr-regulated hyaluronic acid capsule genes (hasABC) by measuring chloramphenicol acetyltransferase (CAT) activity in a reporter strain of group A Streptococcus carrying a has operon promoter-cat fusion. Addition of Mg(2+), but not of Ca(2+), Mn(2+), or Zn(2+), repressed capsule gene expression by up to 80% in a dose-dependent fashion. The decrease in capsule gene transcription was associated with a marked reduction in cell-associated capsular polysaccharide. RNA hybridization analysis demonstrated reduced expression of the Csr-regulated hasABC operon, streptokinase (ska), and streptolysin S (sagA) during growth in the presence of 15 mM Mg(2+) for the wild-type strain 003CAT but not for an isogenic csrS mutant. We propose that Mg(2+) binds to CsrS to induce phosphorylation of CsrR and subsequent repression of virulence gene expression. The low concentration of Mg(2+) in extracellular body fluids predicts that the CsrRCsrS system is maintained in the inactive state during infection, thereby allowing maximal expression of critical virulence determinants in the human host.