O antigen biogenesis sensitises Escherichia coli K-12 to bile salts, providing a plausible explanation for its evolutionary loss.

Escherichia coli K-12 is a model organism for bacteriology and has served as a workhorse for molecular biology and biochemistry for over a century since its first isolation in 1922. However, Escherichia coli K-12 strains are phenotypically devoid of an O antigen (OAg) since early reports in the scientific literature. Recent studies have reported the presence of independent mutations that abolish OAg repeating-unit (RU) biogenesis in E. coli K-12 strains from the same original source, suggesting unknown evolutionary forces have selected for inactivation of OAg biogenesis during the early propagation of K-12. Here, we show for the first time that restoration of OAg in E. coli K-12 strain MG1655 synergistically sensitises bacteria to vancomycin with bile salts (VBS). Suppressor mutants surviving lethal doses of VBS primarily contained disruptions in OAg biogenesis. We present data supporting a model where the transient presence and accumulation of lipid-linked OAg intermediates in the periplasmic leaflet of the inner membrane interfere with peptidoglycan sacculus biosynthesis, causing growth defects that are synergistically enhanced by bile salts. Lastly, we demonstrate that continuous bile salt exposure of OAg-producing MG1655 in the laboratory, can recreate a scenario where OAg disruption is selected for as an evolutionary fitness benefit. Our work thus provides a plausible explanation for the long-held mystery of the selective pressure that may have led to the loss of OAg biogenesis in E. coli K-12; this opens new avenues for exploring long-standing questions on the intricate network coordinating the synthesis of different cell envelope components in Gram-negative bacteria.

Determining glycosyltransferase functional order via lethality due to accumulated O-antigen intermediates, exemplified with Shigella flexneri O-antigen biosynthesis.

The O antigen (OAg) polysaccharide is one of the most diverse surface molecules of Gram-negative bacterial pathogens. The structural classification of OAg, based on serological typing and sequence analysis, is important in epidemiology and the surveillance of outbreaks of bacterial infections. Despite the diverse chemical structures of OAg repeating units (RUs), the genetic basis of RU assembly remains poorly understood and represents a major limitation in assigning gene functions in polysaccharide biosynthesis. Here, we describe a genetic approach to interrogate the functional order of glycosyltransferases (GTs). Using Shigella flexneri as a model, we established an initial glycosyltransferase (IT)-controlled system, which allows functional order allocation of the subsequent GT in a 2-fold manner as follows: (i) first, by reporting the growth defects caused by the sequestration of UndP through disruption of late GTs and (ii) second, by comparing the molecular sizes of stalled OAg intermediates when each putative GT is disrupted. Using this approach, we demonstrate that for RfbF and RfbG, the GT involved in the assembly of S. flexneri backbone OAg RU, RfbG, is responsible for both the committed step of OAg synthesis and the third transferase for the second L-Rha. We also show that RfbF functions as the last GT to complete the S. flexneri OAg RU backbone. We propose that this simple and effective genetic approach can be also extended to define the functional order of enzymatic synthesis of other diverse polysaccharides produced both by Gram-negative and Gram-positive bacteria.IMPORTANCEThe genetic basis of enzymatic assembly of structurally diverse O antigen (OAg) repeating units (RUs) in Gram-negative pathogens is poorly understood, representing a major limitation in our understanding of gene functions for the synthesis of bacterial polysaccharides. We present a simple genetic approach to confidently assign glycosyltransferase (GT) functions and the order in which they act during assembly of the OAg RU. We employed this approach to determine the functional order of GTs involved in Shigella flexneri OAg assembly. This approach can be generally applied in interrogating GT functions encoded by other bacterial polysaccharides to advance our understanding of diverse gene functions in the biosynthesis of polysaccharides, key knowledge in advancing biosynthetic polysaccharide production.

Bladder-draining lymph nodes support germinal center B cell responses during urinary tract infection in mice.

Bacterial urinary tract infections (UTIs) are both common and exhibit high recurrence rates in women. UTI healthcare costs are increasing due to the rise of multidrug-resistant (MDR) bacteria, necessitating alternative approaches for infection control. Here, we directly observed host adaptive immune responses in acute UTI. We employed a mouse model in which wild-type C57BL/6J mice were transurethrally inoculated with a clinically relevant MDR UTI strain of uropathogenic Escherichia coli (UPEC). Firstly, we noted that rag1-/- C57BL/6J mice harbored larger bacterial burdens than wild-type counterparts, consistent with a role for adaptive immunity in UTI control. Consistent with this, UTI triggered in the bladders of wild-type mice early increases of myeloid cells, including CD11chi conventional dendritic cells, suggesting possible involvement of these professional antigen-presenting cells. Importantly, germinal center B cell responses developed by 4 weeks post-infection in bladder-draining lymph nodes of wild-type mice and, although modest in magnitude and transient in nature, could not be boosted with a second UTI. Thus, our data reveal for the first time in a mouse model that UPEC UTI induces local B cell immune responses in bladder-draining lymph nodes, which could potentially serve to control infection.

Simple and Efficient Synthesis of 3-Aryl-2-oxazolidinone Scaffolds Enabling Increased Potency toward Biofilms.

Infectious diseases caused by bacterial pathogens are a leading cause of mortality worldwide. In particular, recalcitrant bacterial communities known as biofilms are implicated in persistent and difficult to treat infections. With a diminishing antibiotic pipeline, new treatments are urgently required to combat biofilm infections. An emerging strategy to develop new treatments is the hybridization of antibiotics. The benefit of this approach is the extension of the useful lifetime of existing antibiotics. The oxazolidinones, which include the last resort antibiotic linezolid, are an attractive target for improving antibiofilm efficacy as they present one of the most recently discovered classes of antibiotics. A key step in the synthesis of new 3-aryl-2-oxazolidinone derivatives is the challenging formation of the oxazolidinone ring. Herein we report a direct synthetic route to the piperazinyl functionalized 3-aryl-2-oxazolidinone 17. We also demonstrate an application of these piperazine molecules by functionalizing them with a nitroxide moiety as a strategy to extend the useful lifetime of oxazolidinones and improve their potency against Methicillin-resistant Staphylococcus aureus (MRSA) biofilms. The antimicrobial susceptibility of the linezolid-nitroxide conjugate 11 and its corresponding methoxyamine derivative 12 (a control for biofilm dispersal) was assessed against planktonic cells and biofilms of MRSA. In comparison to linezolid and our lead compound 10 (a piperazinyl oxazolidinone derivative), the linezolid-nitroxide conjugate 11 displayed a minimum inhibitory concentration that was 4-16-fold higher. The opposite effect was seen in biofilms where the linezolid-nitroxide hybrid 11 was >2-fold more effective (160 µg/mL versus >320 µg/mL) in eradicating MRSA biofilms. The methoxyamine derivative 12 performed on par with linezolid. The drug-likeness of the compounds was also assessed, and all compounds were predicted to have good oral bioavailability. Our piperazinyl oxazolidinone derivative 10 was confirmed to be lead-like and would be a good lead candidate for future functionalized oxazolidinones. The modification of antibiotics with a dispersal agent appears to be a promising approach for eradicating MRSA biofilms and overcoming the antibiotic resistance associated with the biofilm mode of growth.

Thermoresponsive Polymer-Antibiotic Conjugates Based on Gradient Copolymers of 2-Oxazoline and 2-Oxazine.

A polymer-antibiotic conjugate with thermoresponsive properties near body temperature is presented. The backbone polymer is a copolymer of 2-n-propyl-2-oxazine (PropOzi) and methoxycarbonylethyl-2-oxazoline (C2MestOx) which is conjugated with the broad-spectrum antibiotic, cefazolin, via modification of the methyl ester group of C2MestOx. The resulting polymer-antibiotic conjugate has a cloud point temperature near body temperature, meaning that it can form a homogenous solution if cooled, but when injected into a skin-mimic at 37 °C, it forms a drug depot precipitate. Cleavage of the ester linker leads to quantitative release of the pristine cefazolin (with some antibiotic degradation observed) and redissolution of the polymer. When Escherichia coli were treated with polymer-antibiotic conjugate total clearance is observed within 12 h. The power of this approach is the potential for localized antibiotic delivery, for example, at a specific tissue site or into infected phagocytic cells.

Elaboration of a benzofuran scaffold and evaluation of binding affinity and inhibition of Escherichia coli DsbA: A fragment-based drug design approach to novel antivirulence compounds.

Bacterial thiol-disulfide oxidoreductase DsbA is essential for bacterial virulence factor assembly and has been identified as a viable antivirulence target. Herein, we report a structure-based elaboration of a benzofuran hit that bound to the active site groove of Escherichia coli DsbA. Substituted phenyl groups were installed at the 5- and 6-position of the benzofuran using Suzuki-Miyaura coupling. HSQC NMR titration experiments showed dissociation constants of this series in the high µM to low mM range and X-ray crystallography produced three co-structures, showing binding in the hydrophobic groove, comparable with that of the previously reported benzofurans. The 6-(m-methoxy)phenyl analogue (2b), which showed a promising binding pose, was chosen for elaboration from the C-2 position. The 2,6-disubstituted analogues bound to the hydrophobic region of the binding groove and the C-2 groups extended into the more polar, previously un-probed, region of the binding groove. Biochemical analysis of the 2,6-disubsituted analogues showed they inhibited DsbA oxidation activity in vitro. The results indicate the potential to develop the elaborated benzofuran series into a novel class of antivirulence compounds.

Self-derived structure-disrupting peptides targeting methionine aminopeptidase in pathogenic bacteria: a new strategy to generate antimicrobial peptides.

Bacterial infection is one of the leading causes of death in young, elderly, and immune-compromised patients. The rapid spread of multi-drug-resistant (MDR) bacteria is a global health emergency and there is a lack of new drugs to control MDR pathogens. We describe a heretofore-unexplored discovery pathway for novel antibiotics that is based on self-targeting, structure-disrupting peptides. We show that a helical peptide, KFF- EcH3, derived from the Escherichia coli methionine aminopeptidase can disrupt secondary and tertiary structure of this essential enzyme, thereby killing the bacterium (including MDR strains). Significantly, no detectable resistance developed against this peptide. Based on a computational analysis, our study predicted that peptide KFF- EcH3 has the strongest interaction with the structural core of the methionine aminopeptidase. We further used our approach to identify peptide KFF- NgH1 to target the same enzyme from Neisseria gonorrhoeae. This peptide inhibited bacterial growth and was able to treat a gonococcal infection in a human cervical epithelial cell model. These findings present an exciting new paradigm in antibiotic discovery using self-derived peptides that can be developed to target the structures of any essential bacterial proteins.-Zhan, J., Jia, H., Semchenko, E. A., Bian, Y., Zhou, A. M., Li, Z., Yang, Y., Wang, J., Sarkar, S., Totsika, M., Blanchard, H., Jen, F. E.-C., Ye, Q., Haselhorst, T., Jennings, M. P., Seib, K. L., Zhou, Y. Self-derived structure-disrupting peptides targeting methionine aminopeptidase in pathogenic bacteria: a new strategy to generate antimicrobial peptides.

Nitroxide Functionalized Antibiotics Are Promising Eradication Agents against Staphylococcus aureus Biofilms.

Treatment of biofilm-related Staphylococcus aureus infections represents an important medical challenge worldwide, as biofilms, even those involving drug-susceptible S. aureus strains, are highly refractory to conventional antibiotic therapy. Nitroxides were recently shown to induce the dispersal of Gram-negative biofilms in vitro, but their action against Gram-positive bacterial biofilms remains unknown. Here, we demonstrate that the biofilm dispersal activity of nitroxides extends to S. aureus, a clinically important Gram-positive pathogen. Coadministration of the nitroxide CTEMPO (4-carboxy-2,2,6,6-tetramethylpiperidin-1-yloxyl) with ciprofloxacin significantly improved the biofilm eradication activity of the antibiotic against S. aureus Moreover, covalently linking the nitroxide to the antibiotic moiety further reduced the ciprofloxacin minimal biofilm eradication concentration. Microscopy analysis revealed that fluorescent nitroxide-antibiotic hybrids could penetrate S. aureus biofilms and enter cells localized at the surface and base of the biofilm structure. No toxicity to human cells was observed for the nitroxide CTEMPO or the nitroxide-antibiotic hybrids. Taken together, our results show that nitroxides can mediate the dispersal of Gram-positive biofilms and that dual-acting biofilm eradication antibiotics may provide broad-spectrum therapies for the treatment of biofilm-related infections.

Intestinal Colonization Traits of Pandemic Multidrug-Resistant Escherichia coli ST131.

Background: Epidemiological studies point to the gut as a key reservoir of multidrug resistant Escherichia coli multilocus sequence type 131 (ST131), a globally dominant pathogenic clone causing urinary tract and bloodstream infections. Here we report a detailed investigation of its intestinal lifestyle. Methods: Clinical ST131 isolates and type 1 fimbriae null mutants were assessed for colonization of human intestinal epithelia and in mouse intestinal colonization models. Mouse gut tissue underwent histologic analysis for pathology and ST131 localization. Key findings were corroborated in mucus-producing human cell lines and intestinal biopsy specimens. Results: ST131 strains adhered to and invaded human intestinal epithelial cells more than probiotic and commensal strains. The reference ST131 strain EC958 established persistent intestinal colonization in mice, and expression of type 1 fimbriae mediated higher colonization levels. Bacterial loads were highest in the distal parts of the mouse intestine and did not cause any obvious pathology. Further analysis revealed that EC958 could bind to both mucus and underlying human intestinal epithelia. Conclusions: ST131 strains can efficiently colonize the mammalian gut and persist long term. Type 1 fimbriae enhance ST131 intestinal colonization, suggesting that mannosides, currently developed as therapeutics for bladder infections and Crohn's disease, could also be used to limit intestinal ST131 reservoirs.

Comprehensive analysis of type 1 fimbriae regulation in fimB-null strains from the multidrug resistant Escherichia coli ST131 clone.

Uropathogenic Escherichia coli (UPEC) of sequence type 131 (ST131) are a pandemic multidrug resistant clone associated with urinary tract and bloodstream infections. Type 1 fimbriae, a major UPEC virulence factor, are essential for ST131 bladder colonization. The globally dominant sub-lineage of ST131 strains, clade C/H30-R, possess an ISEc55 insertion in the fimB gene that controls phase-variable type 1 fimbriae expression via the invertible fimS promoter. We report that inactivation of fimB in these strains causes altered regulation of type 1 fimbriae expression. Using a novel read-mapping approach based on Illumina sequencing, we demonstrate that 'off' to 'on' fimS inversion is reduced in these strains and controlled by recombinases encoded by the fimE and fimX genes. Unlike typical UPEC strains, the nucleoid-associated H-NS protein does not strongly repress fimE transcription in clade C ST131 strains. Using a genetic screen to identify novel regulators of fimE and fimX in the clade C ST131 strain EC958, we defined a new role for the guaB gene in the regulation of type 1 fimbriae and in colonisation of the mouse bladder. Our results provide a comprehensive analysis of type 1 fimbriae regulation in ST131, and highlight important differences in its control compared to non-ST131 UPEC.

The antigen 43 structure reveals a molecular Velcro-like mechanism of autotransporter-mediated bacterial clumping.

Aggregation and biofilm formation are critical mechanisms for bacterial resistance to host immune factors and antibiotics. Autotransporter (AT) proteins, which represent the largest group of outer-membrane and secreted proteins in Gram-negative bacteria, contribute significantly to these phenotypes. Despite their abundance and role in bacterial pathogenesis, most AT proteins have not been structurally characterized, and there is a paucity of detailed information with regard to their mode of action. Here we report the structure-function relationships of Antigen 43 (Ag43a), a prototypic self-associating AT protein from uropathogenic Escherichia coli. The functional domain of Ag43a displays a twisted L-shaped ß-helical structure firmly stabilized by a 3D hydrogen-bonded scaffold. Notably, the distinctive Ag43a L shape facilitates self-association and cell aggregation. Combining all our data, we define a molecular "Velcro-like" mechanism of AT-mediated bacterial clumping, which can be tailored to fit different bacterial lifestyles such as the formation of biofilms.

Global dissemination of a multidrug resistant Escherichia coli clone.

Escherichia coli sequence type 131 (ST131) is a globally disseminated, multidrug resistant (MDR) clone responsible for a high proportion of urinary tract and bloodstream infections. The rapid emergence and successful spread of E. coli ST131 is strongly associated with several factors, including resistance to fluoroquinolones, high virulence gene content, the possession of the type 1 fimbriae FimH30 allele, and the production of the CTX-M-15 extended spectrum ß-lactamase (ESBL). Here, we used genome sequencing to examine the molecular epidemiology of a collection of E. coli ST131 strains isolated from six distinct geographical locations across the world spanning 2000-2011. The global phylogeny of E. coli ST131, determined from whole-genome sequence data, revealed a single lineage of E. coli ST131 distinct from other extraintestinal E. coli strains within the B2 phylogroup. Three closely related E. coli ST131 sublineages were identified, with little association to geographic origin. The majority of single-nucleotide variants associated with each of the sublineages were due to recombination in regions adjacent to mobile genetic elements (MGEs). The most prevalent sublineage of ST131 strains was characterized by fluoroquinolone resistance, and a distinct virulence factor and MGE profile. Four different variants of the CTX-M ESBL-resistance gene were identified in our ST131 strains, with acquisition of CTX-M-15 representing a defining feature of a discrete but geographically dispersed ST131 sublineage. This study confirms the global dispersal of a single E. coli ST131 clone and demonstrates the role of MGEs and recombination in the evolution of this important MDR pathogen.

Molecular Characterization of the Vacuolating Autotransporter Toxin in Uropathogenic Escherichia coli.

UNLABELLED: The vacuolating autotransporter toxin (Vat) contributes to uropathogenic Escherichia coli (UPEC) fitness during systemic infection. Here, we characterized Vat and investigated its regulation in UPEC. We assessed the prevalence of vat in a collection of 45 UPEC urosepsis strains and showed that it was present in 31 (68%) of the isolates. The isolates containing the vat gene corresponded to three major E. coli sequence types (ST12, ST73, and ST95), and these strains secreted the Vat protein. Further analysis of the vat genomic locus identified a conserved gene located directly downstream of vat that encodes a putative MarR-like transcriptional regulator; we termed this gene vatX The vat-vatX genes were present in the UPEC reference strain CFT073, and reverse transcriptase PCR (RT-PCR) revealed that the two genes are cotranscribed. Overexpression of vatX in CFT073 led to a 3-fold increase in vat gene transcription. The vat promoter region contained three putative nucleation sites for the global transcriptional regulator histone-like nucleoid structuring protein (H-NS); thus, the hns gene was mutated in CFT073 (to generate CFT073 hns). Western blot analysis using a Vat-specific antibody revealed a significant increase in Vat expression in CFT073 hns compared to that in wild-type CFT073. Direct H-NS binding to the vat promoter region was demonstrated using purified H-NS in combination with electrophoresis mobility shift assays. Finally, Vat-specific antibodies were detected in plasma samples from urosepsis patients infected by vat-containing UPEC strains, demonstrating that Vat is expressed during infection. Overall, this study has demonstrated that Vat is a highly prevalent and tightly regulated immunogenic serine protease autotransporter protein of Enterobacteriaceae (SPATE) secreted by UPEC during infection. IMPORTANCE: Uropathogenic Escherichia coli (UPEC) is the major cause of hospital- and community-acquired urinary tract infections. The vacuolating autotransporter toxin (Vat) is a cytotoxin known to contribute to UPEC fitness during murine sepsis infection. In this study, Vat was found to be highly conserved and prevalent among a collection of urosepsis clinical isolates and was expressed at human core body temperature. Regulation of vat was demonstrated to be directly repressed by the global transcriptional regulator H-NS and upregulated by the downstream gene vatX (encoding a new MarR-type transcriptional regulator). Additionally, increased Vat-specific IgG titers were detected in plasma from corresponding urosepsis patients infected with vat-positive isolates. Hence, Vat is a highly conserved and tightly regulated urosepsis-associated virulence factor.

The co-transcriptome of uropathogenic Escherichia coli-infected mouse macrophages reveals new insights into host-pathogen interactions.

Urinary tract infections (UTI) are among the most common infections in humans. Uropathogenic Escherichia coli (UPEC) can invade and replicate within bladder epithelial cells, and some UPEC strains can also survive within macrophages. To understand the UPEC transcriptional programme associated with intramacrophage survival, we performed host-pathogen co-transcriptome analyses using RNA sequencing. Mouse bone marrow-derived macrophages (BMMs) were challenged over a 24 h time course with two UPEC reference strains that possess contrasting intramacrophage phenotypes: UTI89, which survives in BMMs, and 83972, which is killed by BMMs. Neither of these strains caused significant BMM cell death at the low multiplicity of infection that was used in this study. We developed an effective computational framework that simultaneously separated, annotated and quantified the mammalian and bacterial transcriptomes. Bone marrow-derived macrophages responded to the two UPEC strains with a broadly similar gene expression programme. In contrast, the transcriptional responses of the UPEC strains diverged markedly from each other. We identified UTI89 genes up-regulated at 24 h post-infection, and hypothesized that some may contribute to intramacrophage survival. Indeed, we showed that deletion of one such gene (pspA) significantly reduced UTI89 survival within BMMs. Our study provides a technological framework for simultaneously capturing global changes at the transcriptional level in co-cultures, and has generated new insights into the mechanisms that UPEC use to persist within the intramacrophage environment.

The serum resistome of a globally disseminated multidrug resistant uropathogenic Escherichia coli clone.

Escherichia coli ST131 is a globally disseminated, multidrug resistant clone responsible for a high proportion of urinary tract and bloodstream infections. The rapid emergence and successful spread of E. coli ST131 is strongly associated with antibiotic resistance; however, this phenotype alone is unlikely to explain its dominance amongst multidrug resistant uropathogens circulating worldwide in hospitals and the community. Thus, a greater understanding of the molecular mechanisms that underpin the fitness of E. coli ST131 is required. In this study, we employed hyper-saturated transposon mutagenesis in combination with multiplexed transposon directed insertion-site sequencing to define the essential genes required for in vitro growth and the serum resistome (i.e. genes required for resistance to human serum) of E. coli EC958, a representative of the predominant E. coli ST131 clonal lineage. We identified 315 essential genes in E. coli EC958, 231 (73%) of which were also essential in E. coli K-12. The serum resistome comprised 56 genes, the majority of which encode membrane proteins or factors involved in lipopolysaccharide (LPS) biosynthesis. Targeted mutagenesis confirmed a role in serum resistance for 46 (82%) of these genes. The murein lipoprotein Lpp, along with two lipid A-core biosynthesis enzymes WaaP and WaaG, were most strongly associated with serum resistance. While LPS was the main resistance mechanism defined for E. coli EC958 in serum, the enterobacterial common antigen and colanic acid also impacted on this phenotype. Our analysis also identified a novel function for two genes, hyxA and hyxR, as minor regulators of O-antigen chain length. This study offers novel insight into the genetic make-up of E. coli ST131, and provides a framework for future research on E. coli and other Gram-negative pathogens to define their essential gene repertoire and to dissect the molecular mechanisms that enable them to survive in the bloodstream and cause disease.

Molecular analysis of asymptomatic bacteriuria Escherichia coli strain VR50 reveals adaptation to the urinary tract by gene acquisition.

Urinary tract infections (UTIs) are among the most common infectious diseases of humans, with Escherichia coli responsible for >80% of all cases. One extreme of UTI is asymptomatic bacteriuria (ABU), which occurs as an asymptomatic carrier state that resembles commensalism. To understand the evolution and molecular mechanisms that underpin ABU, the genome of the ABU E. coli strain VR50 was sequenced. Analysis of the complete genome indicated that it most resembles E. coli K-12, with the addition of a 94-kb genomic island (GI-VR50-pheV), eight prophages, and multiple plasmids. GI-VR50-pheV has a mosaic structure and contains genes encoding a number of UTI-associated virulence factors, namely, Afa (afimbrial adhesin), two autotransporter proteins (Ag43 and Sat), and aerobactin. We demonstrated that the presence of this island in VR50 confers its ability to colonize the murine bladder, as a VR50 mutant with GI-VR50-pheV deleted was attenuated in a mouse model of UTI in vivo. We established that Afa is the island-encoded factor responsible for this phenotype using two independent deletion (Afa operon and AfaE adhesin) mutants. E. coli VR50afa and VR50afaE displayed significantly decreased ability to adhere to human bladder epithelial cells. In the mouse model of UTI, VR50afa and VR50afaE displayed reduced bladder colonization compared to wild-type VR50, similar to the colonization level of the GI-VR50-pheV mutant. Our study suggests that E. coli VR50 is a commensal-like strain that has acquired fitness factors that facilitate colonization of the human bladder.

Application of fragment-based screening to the design of inhibitors of Escherichia coli DsbA.

The thiol-disulfide oxidoreductase enzyme DsbA catalyzes the formation of disulfide bonds in the periplasm of Gram-negative bacteria. DsbA substrates include proteins involved in bacterial virulence. In the absence of DsbA, many of these proteins do not fold correctly, which renders the bacteria avirulent. Thus DsbA is a critical mediator of virulence and inhibitors may act as antivirulence agents. Biophysical screening has been employed to identify fragments that bind to DsbA from Escherichia coli. Elaboration of one of these fragments produced compounds that inhibit DsbA activity in vitro. In cell-based assays, the compounds inhibit bacterial motility, but have no effect on growth in liquid culture, which is consistent with selective inhibition of DsbA. Crystal structures of inhibitors bound to DsbA indicate that they bind adjacent to the active site. Together, the data suggest that DsbA may be amenable to the development of novel antibacterial compounds that act by inhibiting bacterial virulence.

Sortase A: an ideal target for anti-virulence drug development.

Sortase A is a membrane enzyme responsible for the anchoring of surface-exposed proteins to the cell wall envelope of Gram-positive bacteria. As a well-studied member of the sortase subfamily catalysing the cell wall anchoring of important virulence factors to the surface of staphylococci, enterococci and streptococci, sortase A plays a critical role in Gram-positive bacterial pathogenesis. It is thus considered a promising target for the development of new anti-infective drugs that aim to interfere with important Gram-positive virulence mechanisms, such as adhesion to host tissues, evasion of host defences, and biofilm formation. The additional properties of sortase A as an enzyme that is not required for Gram-positive bacterial growth or viability and is conveniently located on the cell membrane making it more accessible to inhibitor targeting, constitute additional reasons reinforcing the view that sortase A is an ideal target for anti-virulence drug development. Many inhibitors of sortase A have been identified to date using high-throughput or in silico screening of compound libraries (synthetic or natural), and while many have proved useful tools for probing the action model of the enzyme, several are also promising candidates for the development into potent inhibitors. This review is focused on the most promising sortase A inhibitor compounds that are currently in development as leads towards a new class of anti-infective drugs that are urgently needed to help combat the alarming increase in antimicrobial resistance.

Innate transcriptional networks activated in bladder in response to uropathogenic Escherichia coli drive diverse biological pathways and rapid synthesis of IL-10 for defense against bacterial urinary tract infection.

Early transcriptional activation events that occur in bladder immediately following bacterial urinary tract infection (UTI) are not well defined. In this study, we describe the whole bladder transcriptome of uropathogenic Escherichia coli (UPEC) cystitis in mice using genome-wide expression profiling to define the transcriptome of innate immune activation stemming from UPEC colonization of the bladder. Bladder RNA from female C57BL/6 mice, analyzed using 1.0 ST-Affymetrix microarrays, revealed extensive activation of diverse sets of innate immune response genes, including those that encode multiple IL-family members, receptors, metabolic regulators, MAPK activators, and lymphocyte signaling molecules. These were among 1564 genes differentially regulated at 2 h postinfection, highlighting a rapid and broad innate immune response to bladder colonization. Integrative systems-level analyses using InnateDB (http://www.innatedb.com) bioinformatics and ingenuity pathway analysis identified multiple distinct biological pathways in the bladder transcriptome with extensive involvement of lymphocyte signaling, cell cycle alterations, cytoskeletal, and metabolic changes. A key regulator of IL activity identified in the transcriptome was IL-10, which was analyzed functionally to reveal marked exacerbation of cystitis in IL-10-deficient mice. Studies of clinical UTI revealed significantly elevated urinary IL-10 in patients with UPEC cystitis, indicating a role for IL-10 in the innate response to human UTI. The whole bladder transcriptome presented in this work provides new insight into the diversity of innate factors that determine UTI on a genome-wide scale and will be valuable for further data mining. Identification of protective roles for other elements in the transcriptome will provide critical new insight into the complex cascade of events that underpin UTI.