The antimicrobial activity of zinc against group B Streptococcus is strain-dependent across diverse sequence types, capsular serotypes, and invasive versus colonizing isolates.

BACKGROUND: Streptococcus agalactiae or Group B Streptococcus (GBS) is an encapsulated gram-positive bacterial pathobiont that commonly colonizes the lower gastrointestinal tract and reproductive tract of human hosts. This bacterium can infect the gravid reproductive tract and cause invasive infections of pregnant patients and neonates. Upon colonizing the reproductive tract, the bacterial cell is presented with numerous nutritional challenges imposed by the host. One strategy employed by the host innate immune system is intoxication of bacterial invaders with certain transition metals such as zinc. METHODOLOGY: Previous work has demonstrated that GBS must employ elegant strategies to circumnavigate zinc stress in order to survive in the vertebrate host. We assessed 30 strains of GBS from diverse isolation sources, capsular serotypes, and sequence types for susceptibility or resistance to zinc intoxication. RESULTS: Invasive strains, such as those isolated from early onset disease manifestations of GBS infection were significantly less susceptible to zinc toxicity than colonizing strains isolated from rectovaginal swabs of pregnant patients. Additionally, capsular type III (cpsIII) strains and the ST-17 and ST-19 strains exhibited the greatest resilience to zinc stress, whereas ST-1 and ST-12 strains as well as those possessing capsular type Ib (cpsIb) were more sensitive to zinc intoxication. Thus, this study demonstrates that the transition metal zinc possesses antimicrobial properties against a wide range of GBS strains, with isolation source, capsular serotype, and sequence type contributing to susceptibility or resistance to zinc stress.

Simultaneous purification and depolymerization of Streptococcus pneumoniae serotype 2 capsular polysaccharides by trifluoroacetic acid.

Development of an effective purification process in order to provide low cost and high-quality vaccine is the necessity of glycoconjugate vaccine manufacturing industries. In the present study, we have attempted to develop a method for simultaneous purification and depolymerization process for capsular polysaccharides (CPS) derived from Streptococcus pneumoniae serotype 2. Trifluoroacetic acid (TFA) was used to precipitate impurities which were then removed by centrifugation. It was observed that the TFA treatment could simultaneously depolymerize the CPS and purify it. The purified and depolymerized CPS was analyzed for its purity, structural identity and conformity, molecular size, antigenicity to meet desired quality specifications. The obtained results showed that the purification and depolymerization of S. pneumoniae serotype 2 CPS did not affect the antigenicity of CPS.

Revelation of Function and Inhibition of Wza Through Single-Channel Studies.

Antibacterial resistance (AR) is causing more and more bacterial infections that cannot be cured by using the antibacterial drugs that are currently available. It is predicted that 10 million people will die every year by 2050 from infections caused by antibacterial resistant strains, surpassing the predicted numbers of deaths caused by cancer. AR is therefore a global challenge and novel antibacterial strategies are in high demand. To this end, the work on exploring the pore properties of a bacterial sugar transporter, WzaK30, has led to the discovery of the first inhibitor against bacterial capsular polysaccharides export.Recently, single-molecule recapitulation of capsular polysaccharide (CPS) export and pore formation properties of Wza barrel peptides have also revealed the possibility of a next-generation of Wza strategies. These strategies are based upon the first examination and understanding of the pore properties of wild-type (WT) and mutant WzaK30 in single-molecule electrical channel recording. The initially reported experimental procedures have been further developed to enable efficient studies of other Wza homologs that are more common in bacterial pathogens causing significant bacterial infections. Therefore, this chapter presents the most recent protocols and logistics behind the research on Wza channel activity, antibacterials, and strategies. The disciplines covered here include computation, molecular biology, biochemistry, electrophysiology, microbiology, and biophysics.

Rcs Phosphorelay Responses to Truncated Lipopolysaccharide-Induced Cell Envelope Stress in Yersinia enterocolitica.

Lipopolysaccharide (LPS) is the major component of the outer membrane of Gram-negative bacteria, and its integrity is monitored by various stress response systems. Although the Rcs system is involved in the envelope stress response and regulates genes controlling numerous bacterial cell functions of Yersinia enterocolitica, whether it can sense the truncated LPS in Y. enterocolitica remains unclear. In this study, the deletion of the Y. enterocolitica waaF gene truncated the structure of LPS and produced a deep rough LPS. The truncated LPS increased the cell surface hydrophobicity and outer membrane permeability, generating cell envelope stress. The truncated LPS also directly exposed the smooth outer membrane to the external environment and attenuated the resistance to adverse conditions, such as impaired survival under polymyxin B and sodium dodecyl sulfate (SDS) exposure. Further phenotypic experiment and gene expression analysis indicated that the truncated LPS was correlated with the activation of the Rcs phosphorelay, thereby repressing cell motility and biofilm formation. Our findings highlight the importance of LPS integrity in maintaining membrane function and broaden the understanding of Rcs phosphorelay signaling in response to cell envelope stress, thus opening new avenues to develop effective antimicrobial agents for combating Y. enterocolitica infections.

Advantages and limitations of microtiter biofilm assays in the model of antibiofilm activity of Klebsiella phage KP34 and its depolymerase.

One of the potential antibiofilm strategies is to use lytic phages and phage-derived polysaccharide depolymerases. The idea is to uncover bacteria embedded in the biofilm matrix making them accessible and vulnerable to antibacterials and the immune system. Here we present the antibiofilm efficiency of lytic phage KP34 equipped with virion-associated capsule degrading enzyme (depolymerase) and its recombinant depolymerase KP34p57, depolymerase-non-bearing phage KP15, and ciprofloxacin, separately and in combination, using a multidrug-resistant K. pneumoniae biofilm model. The most effective antibiofilm agents were (1) phage KP34 alone or in combination with ciprofloxacin/phage KP15, and (2) depolymerase KP34p57 with phage KP15 and ciprofloxacin. Secondly, applying the commonly used biofilm microtiter assays: (1) colony count, (2) LIVE/DEAD BacLight Bacterial Viability Kit, and (3) crystal violet (CV) biofilm staining, we unravelled the main advantages and limitations of the above methods in antibiofilm testing. The diverse mode of action of selected antimicrobials strongly influenced obtained results, including a false positive enlargement of biofilm mass (CV staining) while applying polysaccharide degrading agents. We suggest that to get a proper picture of antimicrobials' effectiveness, multiple examination methods should be used and the results must be read considering the principle of each technique and the antibacterial mechanism.

Therapeutic Activity of Type 3 Streptococcus pneumoniae Capsule Degrading Enzyme Pn3Pase.

PURPOSE: Streptococcus pneumoniae (Spn) serotype 3 (Spn3) is considered one of the most virulent serotypes with resistance to conventional vaccine and treatment regimens. Pn3Pase is a glycoside hydrolase that we have previously shown to be highly effective in degrading the capsular polysaccharide of type 3 Spn, sensitizing it to host immune clearance. To begin assessing the value and safety of this enzyme for future clinical studies, we investigated the effects of high doses of Pn3Pase on host cells and immune system. METHODS: We assessed the enzyme's catalytic activity following administration in mice, and performed septic infection models to determine if prior administration of the enzyme inhibited repeat treatments of Spn3-challenged mice. We assessed immune populations in mouse tissues following administration of the enzyme, and tested Pn3Pase toxicity on other mammalian cell types in vitro. RESULTS: Repeated administration of the enzyme in vivo does not prevent efficacy of the enzyme in promoting bacterial clearance following bacterial challenge, with insignificant antibody response generated against the enzyme. Immune homeostasis is maintained following high-dose treatment with Pn3Pase, and no cytotoxic effects were observed against mammalian cells. CONCLUSIONS: These data indicate that Pn3Pase has potential as a therapy against Spn3. Further development as a drug product could overcome a great hurdle of pneumococcal infections.

Defining principles that influence antimicrobial peptide activity against capsulated Klebsiella pneumoniae.

The extracellular polysaccharide capsule of Klebsiella pneumoniae resists penetration by antimicrobials and protects the bacteria from the innate immune system. Host antimicrobial peptides are inactivated by the capsule as it impedes their penetration to the bacterial membrane. While the capsule sequesters most peptides, a few antimicrobial peptides have been identified that retain activity against encapsulated K. pneumoniae, suggesting that this bacterial defense can be overcome. However, it is unclear what factors allow peptides to avoid capsule inhibition. To address this, we created a peptide analog with strong antimicrobial activity toward several K. pneumoniae strains from a previously inactive peptide. We characterized the effects of these two peptides on K. pneumoniae, along with their physical interactions with K. pneumoniae capsule. Both peptides disrupted bacterial cell membranes, but only the active peptide displayed this activity against capsulated K. pneumoniae Unexpectedly, the active peptide showed no decrease in capsule binding, but did lose secondary structure in a capsule-dependent fashion compared with the inactive parent peptide. We found that these characteristics are associated with capsule-peptide aggregation, leading to disruption of the K. pneumoniae capsule. Our findings reveal a potential mechanism for disrupting the protective barrier that K. pneumoniae uses to avoid the immune system and last-resort antibiotics.

Oxidative killing of encapsulated and nonencapsulated Streptococcus pneumoniae by lactoperoxidase-generated hypothiocyanite.

Streptococcus pneumoniae (Pneumococcus) infections affect millions of people worldwide, cause serious mortality and represent a major economic burden. Despite recent successes due to pneumococcal vaccination and antibiotic use, Pneumococcus remains a significant medical problem. Airway epithelial cells, the primary responders to pneumococcal infection, orchestrate an extracellular antimicrobial system consisting of lactoperoxidase (LPO), thiocyanate anion and hydrogen peroxide (H2O2). LPO oxidizes thiocyanate using H2O2 into the final product hypothiocyanite that has antimicrobial effects against a wide range of microorganisms. However, hypothiocyanite's effect on Pneumococcus has never been studied. Our aim was to determine whether hypothiocyanite can kill S. pneumoniae. Bactericidal activity was measured in a cell-free in vitro system by determining the number of surviving pneumococci via colony forming units on agar plates, while bacteriostatic activity was assessed by measuring optical density of bacteria in liquid cultures. Our results indicate that hypothiocyanite generated by LPO exerted robust killing of both encapsulated and nonencapsulated pneumococcal strains. Killing of S. pneumoniae by a commercially available hypothiocyanite-generating product was even more pronounced than that achieved with laboratory reagents. Catalase, an H2O2 scavenger, inhibited killing of pneumococcal by hypothiocyanite under all circumstances. Furthermore, the presence of the bacterial capsule or lytA-dependent autolysis had no effect on hypothiocyanite-mediated killing of pneumococci. On the contrary, a pneumococcal mutant deficient in pyruvate oxidase (main bacterial H2O2 source) had enhanced susceptibility to hypothiocyanite compared to its wild-type strain. Overall, results shown here indicate that numerous pneumococcal strains are susceptible to LPO-generated hypothiocyanite.

Anti-Cryptococcal activity of a furanone derivative-antibiofilm and opsonophagocytic potential.

Cryptococcus neoformans, an encapsulated fungal pathogen is evolving as a major threat to immune-compromised patients and rarely to healthy individuals also. The cell wall bound capsular polysaccharide, melanin pigment and biofilm formation are major virulence factors that are known to contribute to cryptococcal meningitis. In the present study, a furanone derivative, (E)-5-benzylidenedihydrofuran-2(3H)-one (compound-6) was evaluated against biofilm of seven different strains of C. neoformans in melanized and non-melanized condition. In addition, the efficacy of compound-6 in activation of TLR-2, opsonophagocytosis, and modulation of cytokine expression during phagocytosis were studied. During the biofilm study, we found that moderate capsule size favored biofilm formation. Interestingly, the minimum biofilm eradication concentration (MBEC0.5) of melanized biofilm was found to be achieved at 1- to 1.7-fold higher MBEC0.5 of non-melanized cells. The maximum eradication of 77% and 69% of non-melanized and melanized biofilm were observed. The capsule size was reduced to half of its size with marked changes in morphology. Furthermore, expression of TLR2, iNOS and pro-inflammatory cytokines such as TNF-α, IL-12, and IFN-γ were also facilitated by compound-6. The correlation analysis showed a positive correlation between phagocytosis and the expression of TLR-2, iNOS, IL-6, IL-12. Collectively, the significant effect of compound-6, anti-melanization activity, antibiofilmand effective immunomodulant could be an interesting dual strategy drug agonist against cryptococcal meningitis.

The Interaction of Klebsiella pneumoniae With Lipid Rafts-Associated Cholesterol Increases Macrophage-Mediated Phagocytosis Due to Down Regulation of the Capsule Polysaccharide.

Klebsiella pneumoniae successfully colonizes host tissues by recognizing and interacting with cholesterol present on membrane-associated lipid rafts. In this study, we evaluated the role of cholesterol in the expression of capsule polysaccharide genes of K. pneumoniae and its implication in resistance to phagocytosis. Our data revealed that exogenous cholesterol added to K. pneumoniae increases macrophage-mediated phagocytosis. To explain this event, the expression of capsular galF, wzi, and manC genes was determined in the presence of cholesterol. Down-regulation of these capsular genes occurred leading to increased susceptibility to phagocytosis by macrophages. In contrast, depletion of cholesterol from macrophage membranes led to enhanced expression of galF, wzi, and manC genes and to capsule production resulting in resistance to macrophage-mediated phagocytosis. Cholesterol-mediated repression of capsular genes was dependent on the RcsA and H-NS global regulators. Finally, cholesterol also down-regulated the expression of genes responsible for LPS core oligosaccharides production and OMPs. Our results suggest that cholesterol plays an important role for the host by reducing the anti-phagocytic properties of the K. pneumoniae capsule facilitating bacterial engulfment by macrophages during the bacteria-eukaryotic cell interaction mediated by lipid rafts.

Molecular epidemiology of invasive and non-invasive group B Streptococcus circulating in Serbia.

Streptococcus agalactiae (group B Streptococcus, GBS) remains the leading cause of invasive diseases in neonates and an important cause of infections in the elderly. The aim of this study was to access the prevalence of GBS genito-rectal colonisation of pregnant women and to evaluate the genetic characteristics of invasive and non-invasive GBS isolates recovered throughout Serbia. A total of 432 GBS isolates were tested for antimicrobial susceptibility, capsular polysaccharide (CPS) types and the presence of the hvgA gene. One hundred one randomly selected isolates were further characterized by clustered regularly interspaced short palindromic repeats (CRISPRs) analysis and/or multilocus sequence typing (MLST). The prevalence of GBS colonization in pregnant women was 15%. Overall, six capsular types (Ia, Ib, II to V) were identified, the most common being III (32.2%) and V (25.2%). The hiper-virulent clone type III/ST17 was present in 43.1% and 6.3% (p < 0.05) of paediatric and adults isolates, respectively. Comparative sequence analysis of the CRISPR1 spacers content indicated that a few clones comprised the vast majority of the tested GBS isolates. Thus, it was estimated that dominant clones recovered from infants were CPS III/ST17 in late-onset infections (19/23; 82.6%), and Ia/ST23 in early-onset disease (44.4%). Conversely, genotype CPS V/ST1 was the most prevalent in adults (4/9; 25.4%). All isolates were susceptible to penicillin. Macrolide resistance (23.1%) was strongly associated with the ermB gene and constitutive resistance to clindamycin (63.9%). The majority of strains was resistant to tetracycline (86.6%), mostly mediated by the tetM gene (87.7%). GBS isolates of CPS V/ST1 and CPS III/ST23 were significantly associated with macrolide and tetracycline resistance, respectively. In conclusion, hyper-virulent CPS III/ST17 and V/ST1 were recognized as dominant GBS clones in this study.

Human serum albumin alters specific genes that can play a role in survival and persistence in Acinetobacter baumannii.

In the past few decades Acinetobacter baumannii has emerged as a notorious nosocomial pathogen because of its ability to acquire genetic material and persist in extreme environments. Recently, human serum albumin (HSA) was shown to significantly increase natural transformation frequency in A. baumannii. This observation led us to perform transcriptomic analysis of strain A118 under HSA induction to identify genes that are altered by HSA. Our results revealed the statistically significant differential expression of 296 protein-coding genes, including those associated with motility, biofilm formation, metabolism, efflux pumps, capsule synthesis, and transcriptional regulation. Phenotypic analysis of these traits showed an increase in surface-associated motility, a decrease in biofilm formation, reduced activity of a citric acid cycle associated enzyme, and increased survival associated with zinc availability. Furthermore, the expression of genes known to play a role in pathogenicity and antibiotic resistance were altered. These genes included those associated with RND-type efflux pumps, the type VI secretion system, iron acquisition/metabolism, and ß-lactam resistance. Together, these results illustrate how human products, in particular HSA, may play a significant role in both survival and persistence of A. baumannii.

Spontaneous point mutations in the capsule synthesis locus leading to structural and functional changes of the capsule in serogroup A meningococcal populations.

Whole genome sequencing analysis of 100 Neisseria meningitidis serogroup A isolates has revealed that the csaABCD-ctrABCD-ctrEF capsule polysaccharide synthesis locus represents a spontaneous point mutation hotspot. Structural and functional properties of the capsule of 11 carriage and two disease isolates with non-synonymous point mutations or stop codons in capsule synthesis genes were analyzed for their capsular polysaccharide expression, recognition by antibodies and sensitivity to bactericidal killing. Eight of eleven carriage isolates presenting capsule locus mutations expressed no or reduced amounts of capsule. One isolate with a stop codon in the O-acetyltransferase gene expressed non-O-acetylated polysaccharide, and was not recognized by anti-capsule antibodies. Capsule and O-acetylation deficient mutants were resistant to complement deposition and killing mediated by anti-capsular antibodies, but not by anti-lipopolysaccharide antibodies. Two capsule polymerase mutants, one carriage and one case isolate, showed capsule over-expression and increased resistance against bactericidal activity of both capsule- and lipopolysaccharide-specific antibodies. Meningococci have developed multiple strategies for changing capsule expression and structure, which is relevant both for colonization and virulence. Here we show that point mutations in the capsule synthesis genes substantially contribute to the repertoire of genetic mechanisms in natural populations leading to variability in capsule expression.

Galectin-3 impacts Cryptococcus neoformans infection through direct antifungal effects.

Cryptococcus neoformans is an encapsulated fungal pathogen that causes cryptococcosis, which is a major opportunistic infection in immunosuppressed individuals. Mammalian ß-galactoside-binding protein Galectin-3 (Gal-3) modulates the host innate and adaptive immunity, and plays significant roles during microbial infections including some fungal diseases. Here we show that this protein plays a role also in C. neoformans infection. We find augmented Gal-3 serum levels in human and experimental infections, as well as in spleen, lung, and brain tissues of infected mice. Gal-3-deficient mice are more susceptible to cryptococcosis than WT animals, as demonstrated by the higher fungal burden and lower animal survival. In vitro experiments show that Gal-3 inhibits fungal growth and exerts a direct lytic effect on C. neoformans extracellular vesicles (EVs). Our results indicate a direct role for Gal-3 in antifungal immunity whereby this molecule affects the outcome of C. neoformans infection by inhibiting fungal growth and reducing EV stability, which in turn could benefit the host.

CRH Affects the Phenotypic Expression of Sepsis-Associated Virulence Factors by Streptococcus pneumoniae Serotype 1 In vitro.

Sepsis is a life-threatening health condition caused by infectious pathogens of the respiratory tract, and accounts for 28-50% of annual deaths in the US alone. Current treatment regimen advocates the use of corticosteroids as adjunct treatment with antibiotics, for their broad inhibitory effect on the activity and production of pro-inflammatory mediators. However, despite their use, corticosteroids have not proven to be able to reverse the death incidence among septic patients. We have previously demonstrated the potential for neuroendocrine factors to directly influence Streptococcus pneumoniae virulence, which may in turn mediate disease outcome leading to sepsis and septic shock. The current study investigated the role of Corticotropin-releasing hormone (CRH) in mediating key markers of pneumococcal virulence as important phenotypic determinants of sepsis and septic shock risks. In vitro cultures of serotype 1 pneumococcal strain with CRH promoted growth rate, increased capsule thickness and penicillin resistance, as well as induced pneumolysin gene expression. These results thus provide significant insights of CRH-pathogen interactions useful in understanding the underlying mechanisms of neuroendocrine factor's role in the onset of community acquired pneumonias (CAP), sepsis and septic shock.

A nanomechanical study of the effects of colistin on the Klebsiella pneumoniae AJ218 capsule.

Atomic force microscopy measurements of capsule thickness revealed that that the wild-type Klebsiella pneumoniae AJ218 capsular polysaccharides were rearranged by exposure to colistin. The increase in capsule thickness measured near minimum inhibitory/bactericidal concentration (MIC/MBC) is consistent with the idea that colistin displaces the divalent cations that cross-bridge adjacent lipopolysaccharide (LPS) molecules through the capsule network. Cryo-electron microscopy demonstrated that the measured capsule thickness at near MIC/MBC of 1.2 µM was inflated by the disrupted outer membrane, through which the capsule is excreted and LPS is bound. Since wild-type and capsule-deficient strains of K. pneumoniae AJ218 have equivalent MICs and MBCs, the presence of the capsule appeared to confer no protection against colistin in AJ218. A spontaneously arising colistin mutant showed a tenfold increase in resistance to colistin; genetic analysis identified a single amino acid substitution (Q95P) in the PmrB sensor kinase in this colistin-resistant K. pneumoniae AJ218. Modification of the lipid A component of the LPS could result in a reduction of the net-negative charge of the outer membrane, which could hinder binding of colistin to the outer membrane and displacement of the divalent cations that bridge adjacent LPS molecules throughout the capsular polysaccharide network. Retention of the cross-linking divalent cations may explain why measurements of capsule thickness did not change significantly in the colistin-resistant strain after colistin exposure. These results contrast with those for other K. pneumoniae strains that suggest that the capsule confers colistin resistance.

Capsular polysaccharide production and serum survival of Vibrio vulnificus are dependent on antitermination control by RfaH.

The human pathogen Vibrio vulnificus undergoes phase variation among colonial morphotypes, including a virulent opaque form which produces capsular polysaccharide (CPS) and a translucent phenotype that produces little or no CPS and is attenuated. Here, we found that a V. vulnificus mutant defective for RfaH antitermination control showed a diminished capacity to undergo phase variation and displayed significantly reduced distal gene expression within the Group I CPS operon. Moreover, the rfaH mutant produced negligible CPS and was highly sensitive to killing by normal human serum, results which indicate that RfaH is likely essential for virulence in this bacterium.

Impact of glycemic control on capsular polysaccharide biosynthesis and opsonophagocytosis of Klebsiella pneumoniae: Implications for invasive syndrome in patients with diabetes mellitus.

Klebsiella pneumoniae (KP), with production of abundant capsular polysaccharide (CPS), is capable of causing invasive syndrome. Environmental glucose stimuli may increase CPS biosynthesis. We aimed to investigate the relationship between glycemic control and KP-mediated invasive syndrome in diabetic patients and the effect of glucose on CPS biosynthesis. Diabetic patients with community-acquired KP bacteremia were included to study the risk factors of invasive syndrome. KP-M1, a serotype-K1 KP clinical isolate, was used to examine the CPS biosynthesis and cps gene expression, and the effect of exogenous glucose on bacterial phagocytosis and killing. We found that invasive syndrome was significantly more common in diabetic patients who were infected with strains expressing the K1 serotype (adjusted odds ratio [AOR], 8.32; 95% confidence interval [CI], 1.56-44.24; p=0.01), and had poor glycemic control (HbA1c ≥9%; AOR, 5.66; 95% CI, 2.01-15.92; p<0.01). Pre-incubation of KP-M1 in media containing different gradient glucose concentrations enhanced CPS biosynthesis and cps gene expression in high glucose (0.5%) concentration, which leads to increasing bacterial resistance to phagocytosis and killing. High glucose levels reflected by poor glycemic control may stimulate CPS biosynthesis and cps gene expression of highly virulent KP, which increase resistance to phagocytosis and contribute to development of invasive syndrome.

Inorganic Phosphate Limitation Modulates Capsular Polysaccharide Composition in Mycobacteria.

Mycobacterium tuberculosis is protected by an unusual and highly impermeable cell envelope that is critically important for the successful colonization of the host. The outermost surface of this cell envelope is formed by capsular polysaccharides that play an important role in modulating the initial interactions once the bacillus enters the body. Although the bioenzymatic steps involved in the production of the capsular polysaccharides are emerging, information regarding the ability of the bacterium to modulate the composition of the capsule is still unknown. Here, we study the mechanisms involved in regulation of mycobacterial capsule biosynthesis using a high throughput screen for gene products involved in capsular α-glucan production. Utilizing this approach we identified a group of mutants that all carried mutations in the ATP-binding cassette phosphate transport locus pst These mutants collectively exhibited a strong overproduction of capsular polysaccharides, including α-glucan and arabinomannan, suggestive of a role for inorganic phosphate (Pi) metabolism in modulating capsular polysaccharide production. These findings were corroborated by the observation that growth under low Pi conditions as well as chemical activation of the stringent response induces capsule production in a number of mycobacterial species. This induction is, in part, dependent on σ factor E. Finally, we show that Mycobacterium marinum, a model organism for M. tuberculosis, encounters Pi stress during infection, which shows the relevance of our findings in vivo.

Threading the Needle: Small-Molecule Targeting of a Xenobiotic Receptor to Ablate Escherichia coli Polysaccharide Capsule Expression Without Altering Antibiotic Resistance.

BACKGROUND: Uropathogenic Escherichia coli (UPEC), a leading cause of urinary tract and invasive infections worldwide, is rapidly acquiring multidrug resistance, hastening the need for selective new anti-infective agents. Here we demonstrate the molecular target of DU011, our previously discovered potent, nontoxic, small-molecule inhibitor of UPEC polysaccharide capsule biogenesis and virulence. METHODS: Real-time polymerase chain reaction analysis and a target-overexpression drug-suppressor screen were used to localize the putative inhibitor target. A thermal shift assay quantified interactions between the target protein and the inhibitor, and a novel DNase protection assay measured chemical inhibition of protein-DNA interactions. Virulence of a regulatory target mutant was assessed in a murine sepsis model. RESULTS: MprA, a MarR family transcriptional repressor, was identified as the putative target of the DU011 inhibitor. Thermal shift measurements indicated the formation of a stable DU011-MprA complex, and DU011 abrogated MprA binding to its DNA promoter site. Knockout of mprA had effects similar to that of DU011 treatment of wild-type bacteria: a loss of encapsulation and complete attenuation in a murine sepsis model, without any negative change in antibiotic resistance. CONCLUSIONS: MprA regulates UPEC polysaccharide encapsulation, is essential for UPEC virulence, and can be targeted without inducing antibiotic resistance.