Polysaccharide-Targeting Lipid Nanoparticles to Kill Gram-Negative Bacteria.

The rapid increase and spread of Gram-negative bacteria resistant to many or all existing treatments threaten a return to the preantibiotic era. The presence of bacterial polysaccharides that impede the penetration of many antimicrobials and protect them from the innate immune system contributes to resistance and pathogenicity. No currently approved antibiotics target the polysaccharide regions of microbes. Here, describe monolaurin-based niosomes, the first lipid nanoparticles that can eliminate bacterial polysaccharides from hypervirulent Klebsiella pneumoniae, are described. Their combination with polymyxin B shows no cytotoxicity in vitro and is highly effective in combating K. pneumoniae infection in vivo. Comprehensive mechanistic studies have revealed that antimicrobial activity proceeds via a multimodal mechanism. Initially, lipid nanoparticles disrupt polysaccharides, then outer and inner membranes are destabilized and destroyed by polymyxin B, resulting in synergistic cell lysis. This novel lipidic nanoparticle system shows tremendous promise as a highly effective antimicrobial treatment targeting multidrug-resistant Gram-negative pathogens.

Vaccine-induced inflammation and inflammatory monocytes promote CD4+ T cell-dependent immunity against murine salmonellosis.

Prior infection can generate protective immunity against subsequent infection, although the efficacy of such immunity can vary considerably. Live-attenuated vaccines (LAVs) are one of the most effective methods for mimicking this natural process, and analysis of their efficacy has proven instrumental in the identification of protective immune mechanisms. Here, we address the question of what makes a LAV efficacious by characterising immune responses to a LAV, termed TAS2010, which is highly protective (80-90%) against lethal murine salmonellosis, in comparison with a moderately protective (40-50%) LAV, BRD509. Mice vaccinated with TAS2010 developed immunity systemically and were protected against gut-associated virulent infection in a CD4+ T cell-dependent manner. TAS2010-vaccinated mice showed increased activation of Th1 responses compared with their BRD509-vaccinated counterparts, leading to increased Th1 memory populations in both lymphoid and non-lymphoid organs. The optimal development of Th1-driven immunity was closely correlated with the activation of CD11b+Ly6GnegLy6Chi inflammatory monocytes (IMs), the activation of which can be modulated proportionally by bacterial load in vivo. Upon vaccination with the LAV, IMs expressed T cell chemoattractant CXCL9 that attracted CD4+ T cells to the foci of infection, where IMs also served as a potent source of antigen presentation and Th1-promoting cytokine IL-12. The expression of MHC-II in IMs was rapidly upregulated following vaccination and then maintained at an elevated level in immune mice, suggesting IMs may have a role in sustained antigen stimulation. Our findings present a longitudinal analysis of CD4+ T cell development post-vaccination with an intracellular bacterial LAV, and highlight the benefit of inflammation in the development of Th1 immunity. Future studies focusing on the induction of IMs may reveal key strategies for improving vaccine-induced T cell immunity.

The evolutionary mechanism of non-carbapenemase carbapenem-resistant phenotypes in Klebsiella spp.

Antibiotic resistance is driven by selection, but the degree to which a bacterial strain's evolutionary history shapes the mechanism and strength of resistance remains an open question. Here, we reconstruct the genetic and evolutionary mechanisms of carbapenem resistance in a clinical isolate of Klebsiella quasipneumoniae. A combination of short- and long-read sequencing, machine learning, and genetic and enzymatic analyses established that this carbapenem-resistant strain carries no carbapenemase-encoding genes. Genetic reconstruction of the resistance phenotype confirmed that two distinct genetic loci are necessary in order for the strain to acquire carbapenem resistance. Experimental evolution of the carbapenem-resistant strains in growth conditions without the antibiotic revealed that both loci confer a significant cost and are readily lost by de novo mutations resulting in the rapid evolution of a carbapenem-sensitive phenotype. To explain how carbapenem resistance evolves via multiple, low-fitness single-locus intermediates, we hypothesised that one of these loci had previously conferred adaptation to another antibiotic. Fitness assays in a range of drug concentrations show how selection in the antibiotic ceftazidime can select for one gene (blaDHA-1) potentiating the evolution of carbapenem resistance by a single mutation in a second gene (ompK36). These results show how a patient's treatment history might shape the evolution of antibiotic resistance and could explain the genetic basis of carbapenem-resistance found in many enteric-pathogens.

Functional flexibility and plasticity in immune control of systemic Salmonella infection.

Immunity to systemic Salmonella infection depends on multiple effector mechanisms. Lymphocyte-derived interferon gamma (IFN-γ) enhances cell-intrinsic bactericidal capabilities to antagonize the hijacking of phagocytes as replicative niches for Salmonella. Programmed cell death (PCD) provides another means through which phagocytes fight against intracellular Salmonella. We describe remarkable levels of flexibility with which the host coordinates and adapts these responses. This involves interchangeable cellular sources of IFN-γ regulated by innate and adaptive cues, and the rewiring of PCD pathways in previously unknown ways. We discuss that such plasticity is likely the consequence of host-pathogen coevolution and raise the possibility of further functional overlap between these seemingly distinct processes.

LPS O Antigen Plays a Key Role in Klebsiella pneumoniae Capsule Retention.

Despite the importance of encapsulation in bacterial pathogenesis, the biochemical mechanisms and forces that underpin retention of capsule by encapsulated bacteria are poorly understood. In Gram-negative bacteria, there may be interactions between lipopolysaccharide (LPS) core and capsule polymers, between capsule polymers with retained acyl carriers and the outer membrane, and in some bacteria, between the capsule polymers and Wzi, an outer membrane protein lectin. Our transposon studies in Klebsiella pneumoniae B5055 identified additional genes that, when insertionally inactivated, resulted in reduced encapsulation. Inactivation of the gene waaL, which encodes the ligase responsible for attaching the repeated O antigen of LPS to the LPS core, resulted in a significant reduction in capsule retention, measured by atomic force microscopy. This reduction in encapsulation was associated with increased sensitivity to human serum and decreased virulence in a murine model of respiratory infection and, paradoxically, with increased biofilm formation. The capsule in the WaaL mutant was physically smaller than that of the Wzi mutant of K. pneumoniae B5055. These results suggest that interactions between surface carbohydrate polymers may enhance encapsulation, a key phenotype in bacterial virulence, and provide another target for the development of antimicrobials that may avoid resistance issues associated with growth inhibition. IMPORTANCE Bacterial capsules, typically comprised of complex sugars, enable pathogens to avoid key host responses to infection, including phagocytosis. These capsules are synthesized within the bacteria, exported through the outer envelope, and then secured to the external surface of the organism by a force or forces that are incompletely described. This study shows that in the important hospital pathogen Klebsiella pneumoniae, the polysaccharide capsule is retained by interactions with other surface sugars, especially the repeated sugar molecule of the LPS molecule in Gram-negative bacteria known as "O antigen." This O antigen is joined to the LPS molecule by ligation, and loss of the enzyme responsible for ligation, a protein called WaaL, results in reduced encapsulation. Since capsules are essential to the virulence of many pathogens, WaaL might provide a target for new antimicrobial development, critical to the control of pathogens like K. pneumoniae that have become highly drug resistant.

Genomic epidemiology of Salmonella Typhi in Central Division, Fiji, 2012 to 2016.

Background: Typhoid fever is endemic in some Pacific Island Countries including Fiji and Samoa yet genomic surveillance is not routine in such settings. Previous studies suggested imports of the global H58 clade of Salmonella enterica var Typhi (Salmonella Typhi) contribute to disease in these countries which, given the MDR potential of H58, does not auger well for treatment. The objective of the study was to define the genomic epidemiology of Salmonella Typhi in Fiji. Methods: Genomic sequencing approaches were implemented to study the distribution of 255 Salmonella Typhi isolates from the Central Division of Fiji. We augmented epidemiological surveillance and Bayesian phylogenomic approaches with a multi-year typhoid case-control study to define geospatial patterns among typhoid cases. Findings: Genomic analyses showed Salmonella Typhi from Fiji resolved into 2 non-H58 genotypes with isolates from the two dominant ethnic groups, the Indigenous (iTaukei) and non-iTaukei genetically indistinguishable. Low rates of international importation of clones was observed and overall, there were very low levels an antibiotic resistance within the endemic Fijian typhoid genotypes. Genomic epidemiological investigations were able to identify previously unlinked case clusters. Bayesian phylodynamic analyses suggested that genomic variation within the larger endemic Salmonella Typhi genotype expanded at discreet times, then contracted. Interpretation: Cyclones and flooding drove 'waves' of typhoid outbreaks in Fiji which, through population aggregation, poor sanitation and water safety, and then mobility of the population, spread clones more widely. Minimal international importations of new typhoid clones suggest that targeted local intervention strategies may be useful in controlling endemic typhoid infection. These findings add to our understanding of typhoid transmission networks in an endemic island country with broad implications, particularly across Pacific Island Countries. Funding: This work was supported by the Coalition Against Typhoid through the Bill and Melinda Gates Foundation [grant number OPP1017518], the Victorian Government, the National Health and Medical Research Council Australia, the Australian Research Council, and the Fiji Ministry of Health and Medical Services.

Genomic dissection of Klebsiella pneumoniae infections in hospital patients reveals insights into an opportunistic pathogen.

Klebsiella pneumoniae is a major cause of opportunistic healthcare-associated infections, which are increasingly complicated by the presence of extended-spectrum beta-lactamases (ESBLs) and carbapenem resistance. We conducted a year-long prospective surveillance study of K. pneumoniae clinical isolates in hospital patients. Whole-genome sequence (WGS) data reveals a diverse pathogen population, including other species within the K. pneumoniae species complex (18%). Several infections were caused by K. variicola/K. pneumoniae hybrids, one of which shows evidence of nosocomial transmission. A wide range of antimicrobial resistance (AMR) phenotypes are observed, and diverse genetic mechanisms identified (mainly plasmid-borne genes). ESBLs are correlated with presence of other acquired AMR genes (median n = 10). Bacterial genomic features associated with nosocomial onset are ESBLs (OR 2.34, p = 0.015) and rhamnose-positive capsules (OR 3.12, p < 0.001). Virulence plasmid-encoded features (aerobactin, hypermucoidy) are observed at low-prevalence (<3%), mostly in community-onset cases. WGS-confirmed nosocomial transmission is implicated in just 10% of cases, but strongly associated with ESBLs (OR 21, p < 1 × 10-11). We estimate 28% risk of onward nosocomial transmission for ESBL-positive strains vs 1.7% for ESBL-negative strains. These data indicate that K. pneumoniae infections in hospitalised patients are due largely to opportunistic infections with diverse strains, with an additional burden from nosocomially-transmitted AMR strains and community-acquired hypervirulent strains.

The Role of ZntA in Klebsiella pneumoniae Zinc Homeostasis.

Klebsiella pneumoniae is an opportunistic Gram-negative pathogen that is a leading cause of healthcare-associated infections, including pneumonia, urinary tract infections, and sepsis. Essential to the colonization and infection by K. pneumoniae is the acquisition of nutrients, such as the transition metal ion zinc. Zinc has crucial structural and catalytic roles in the proteome of all organisms. Nevertheless, in excess, it has the potential to mediate significant toxicity by dysregulating the homeostasis of other transition elements, disrupting enzymatic processes, and perturbing metalloprotein cofactor acquisition. Here, we sought to elucidate the zinc detoxification mechanisms of K. pneumoniae, which remain poorly defined. Using the representative K. pneumoniae AJ218 strain, we showed that the P-type ATPase, ZntA, which is upregulated in response to cellular zinc stress, was the primary zinc efflux pathway. Deletion of zntA rendered K. pneumoniae AJ218 highly susceptible to exogenous zinc stress and manifested as an impaired growth phenotype and increased cellular accumulation of the metal. Loss of zntA also increased sensitivity to cadmium stress, indicating a role for this efflux pathway in cadmium resistance. Disruption of zinc homeostasis in the K. pneumoniae AJ218 ΔzntA strain also impacted manganese and iron homeostasis and was associated with increased production of biofilm. Collectively, this work showed the critical role of ZntA in K. pneumoniae zinc tolerance and provided a foundation for further studies on zinc homeostasis and the future development of novel antimicrobials to target this pathway. IMPORTANCE Klebsiella pneumoniae is a leading cause of healthcare-associated infections, including pneumonia, urinary tract infections, and sepsis. Treatment of K. pneumoniae infections is becoming increasingly challenging due to high levels of antibiotic resistance and the rising prevalence of carbapenem-resistant, extended-spectrum ß-lactamases producing strains. Zinc is essential to the colonization and infection by many bacterial pathogens but toxic in excess. This work described the first dissection of the pathways associated with resisting extracellular zinc stress in K. pneumoniae. This study revealed that the P-type ATPase ZntA was highly upregulated in response to exogenous zinc stress and played a major role in maintaining bacterial metal homeostasis. Knowledge of how this major bacterial pathogen resists zinc stress provided a foundation for antimicrobial development studies to target and abrogate their essential function.

When secretion turns into excretion - the different roles of IgA.

IgA deficiency is the commonest immunodeficiency affecting up to 1 in 700 individuals. The effects of IgA deficiency are difficult to see in many individuals, are mild in many fewer and severe in fewer still. While monovalent IgA is found in serum, dimeric IgA is secreted through mucosal surfaces where it helps to maintain epithelial homeostasis. Studies with knockout mice have taught us that there are subtle inflammatory consequences of removing secretory IgA (sIgA), and the best explanation for these changes can be related by the loss of the 'excretory' immune system. The excretion of antigens is a logical process in regulating the immune system, given the long half-life of complement fixing antibodies. But the function of IgA as an immune or inflammation regulator may go beyond antigen removal.

Assessment of Rapid Diagnostic Tests for Typhoid Diagnosis and Assessment of Febrile Illness Outbreaks in Fiji.

Typhoid is an endemic in Fiji with increases observed since the early 2000s and frequent outbreaks reported. We assessed the diagnostic accuracy of currently available typhoid rapid diagnostic tests (RDTs) (TUBEX, Typhidot Rapid, and Test-It assay) to establish their performance against blood culture in Fiji and to examine their suitability for rapid typhoid outbreak identification. The performance of RDTs was assessed in the public health reference laboratory in Suva, Fiji, according to the manufacturers' instructions. A simulation was used to examine the potential use of RDTs for attribution of a febrile illness outbreak to typhoid. For the diagnostic evaluation, 179 patients were included; 49 had blood culture-confirmed typhoid, 76 had fever as a result of non-typhoid etiologies, and 54 were age-matched community controls. The median (interquartile range) age was 29 (20-46) years. Of the participants, 92 (51.4%) were male and 131 (73.2%) were indigenous Fijians. The sensitivities of the tests were 77.6% for TUBEX, 75.5% for Typhidot Rapid, and 57.1% for Test-It assay. The Test-It assay had the highest specificity of 93.4%, followed by Typhidot Rapid 85.5% and TUBEX 60.5%. Typhidot Rapid had the best performance in the simulation for attribution of a febrile illness outbreak to typhoid. Typhoid RDTs performed suboptimally for individual patient diagnosis due to low sensitivity and variable specificity. We demonstrate that RDTs could be useful in the field for rapid attribution of febrile illness outbreaks to typhoid. Typhidot Rapid had the best combination of sensitivity, specificity, positive and negative predictive values, cost, and ease of use for this purpose.

CD4+ T cell immunity to Salmonella is transient in the circulation.

While Salmonella enterica is seen as an archetypal facultative intracellular bacterial pathogen where protection is mediated by CD4+ T cells, identifying circulating protective cells has proved very difficult, inhibiting steps to identify key antigen specificities. Exploiting a mouse model of vaccination, we show that the spleens of C57BL/6 mice vaccinated with live-attenuated Salmonella serovar Typhimurium (S. Typhimurium) strains carried a pool of IFN-γ+ CD4+ T cells that could adoptively transfer protection, but only transiently. Circulating Salmonella-reactive CD4+ T cells expressed the liver-homing chemokine receptor CXCR6, accumulated over time in the liver and assumed phenotypic characteristics associated with tissue-associated T cells. Liver memory CD4+ T cells showed TCR selection bias and their accumulation in the liver could be inhibited by blocking CXCL16. These data showed that the circulation of CD4+ T cells mediating immunity to Salmonella is limited to a brief window after which Salmonella-specific CD4+ T cells migrate to peripheral tissues. Our observations highlight the importance of triggering tissue-specific immunity against systemic infections.

Mechanistic Insights into the Capsule-Targeting Depolymerase from a Klebsiella pneumoniae Bacteriophage.

The production of capsular polysaccharides by Klebsiella pneumoniae protects the bacterial cell from harmful environmental factors such as antimicrobial compounds and infection by bacteriophages (phages). To bypass this protective barrier, some phages encode polysaccharide-degrading enzymes referred to as depolymerases to provide access to cell surface receptors. Here, we characterized the phage RAD2, which infects K. pneumoniae strains that produce the widespread, hypervirulence-associated K2-type capsular polysaccharide. Using transposon-directed insertion sequencing, we have shown that the production of capsule is an absolute requirement for efficient RAD2 infection by serving as a first-stage receptor. We have identified the depolymerase responsible for recognition and degradation of the capsule, determined that the depolymerase forms globular appendages on the phage virion tail tip, and present the cryo-electron microscopy structure of the RAD2 capsule depolymerase at 2.7-Å resolution. A putative active site for the enzyme was identified, comprising clustered negatively charged residues that could facilitate the hydrolysis of target polysaccharides. Enzymatic assays coupled with mass spectrometric analyses of digested oligosaccharide products provided further mechanistic insight into the hydrolase activity of the enzyme, which, when incubated with K. pneumoniae, removes the capsule and sensitizes the cells to serum-induced killing. Overall, these findings expand our understanding of how phages target the Klebsiella capsule for infection, providing a framework for the use of depolymerases as antivirulence agents against this medically important pathogen. IMPORTANCE Klebsiella pneumoniae is a medically important pathogen that produces a thick protective capsule that is essential for pathogenicity. Phages are natural predators of bacteria, and many encode diverse "capsule depolymerases" which specifically degrade the capsule of their hosts, an exploitable trait for potential therapies. We have determined the first structure of a depolymerase that targets the clinically relevant K2 capsule and have identified its putative active site, providing hints to its mechanism of action. We also show that Klebsiella cells treated with a recombinant form of the depolymerase are stripped of capsule, inhibiting their ability to grow in the presence of serum, demonstrating the anti-infective potential of these robust and readily producible enzymes against encapsulated bacterial pathogens such as K. pneumoniae.

CCL17-expressing dendritic cells in the intestine are preferentially infected by Salmonella but CCL17 plays a redundant role in systemic dissemination.

INTRODUCTION: Salmonella spp. are a recognized and global cause of serious health issues from gastroenteritis to invasive disease. The mouse model of human typhoid fever, which uses Salmonella enterica serovar Typhimurium (STM) in susceptible mouse strains, has revealed that the bacteria gain access to extraintestinal tissues from the gastrointestinal tract to cause severe systemic disease. Previous analysis of the immune responses against Salmonella spp. revealed the crucial role played by dendritic cells (DCs) in carrying STM from the intestinal mucosa to the mesenteric lymph nodes (mLNs), a key site for antigen presentation and T cell activation. In this study, we investigated the influence of chemokine CCL17 on the dissemination of STM. METHODS: WT, CCL17/EGFP reporter, or CCL17-deficient mice were infected orally with STM (SL1344) or mCherry-expressing STM for 1-3 days. Colocalization of STM with CCL17-expressing DCs in Peyer's patches (PP) and mLN was analyzed by fluorescence microscopy. In addition, DCs and myeloid cell populations from naïve and Salmonella-infected mice were analyzed by flow cytometry. Bacterial load was determined in PP, mLN, spleen, and liver 1 and 3 days after infection. RESULTS: Histological analysis revealed that CCL17-expressing cells are located in close proximity to STM in the dome area of PP. We show that, in mLN, STM were preferentially located within CCL17+ rather than CCL17- DCs, besides other mononuclear phagocytes, and identified the CD103+ CD11b- DC subset as the main STM-carrying DC population in the intestine. STM infection triggered upregulation of CCL17 expression in specific intestinal DC subsets in a tissue-specific manner. The dissemination of STM from the gut to the mLN, however, was only moderately influenced by the presence of CCL17. CONCLUSION: CCL17-expressing DCs were preferentially infected by Salmonella in the intestine in comparison to other DC. Nevertheless, the production of CCL17 was not essential for the early dissemination of Salmonella from the gut to systemic organs.

The Potential Use of Hypochlorous Acid and a Smart Prefabricated Sanitising Chamber to Reduce Occupation-Related COVID-19 Exposure.

This work is part of a project on the development of a smart prefabricated sanitising chamber (SPSC) to provide extra measures against the transmission of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Stabilised hypochlorous acid (HOCl) is an approved disinfectant against SARS-CoV-2 by the Environmental Protection Association US in its liquid form on non-porous surfaces. This review is extended to cover its viricidal/bactericidal efficacy in aerosolised or sprayed form which showed an effective dose of as low as 20 ppm and the exposure duration of at least 60 s. The aerosolised application was also recommended with particle size of less than 200 µm to increase the contact with pathogens. The review also includes the safety and toxicity of HOCl with different concentrations. The review calls for more investigations into the effect of HOCl in mist and fog form on the respiratory system when transitioning through the proposed SPSC.

Measurement of the interconnected turgor pressure and envelope elasticity of live bacterial cells.

Turgor pressure and envelope elasticity of bacterial cells are two mechanical parameters that play a dominant role in cellular deformation, division, and motility. However, a clear understanding of these two properties is lacking because of their strongly interconnected mechanisms. This study established a nanoindentation method to precisely measure the turgor pressure and envelope elasticity of live bacteria. The indentation force-depth curves of Klebsiella pneumoniae bacteria were recorded with atomic force microscopy. Through combination of dimensional analysis and numerical simulations, an explicit expression was derived to decouple the two properties of individual bacteria from the nanoindentation curves. We show that the Young's modulus of bacterial envelope is sensitive to the external osmotic environment, and the turgor pressure is significantly dependent on the external osmotic stress. This method can not only quantify the turgor pressure and envelope elasticity of bacteria, but also help resolve the mechanical behaviors of bacteria in different environments.

Bacterial Redox Potential Powers Controlled Radical Polymerization.

Microbes employ a remarkably intricate electron transport system to extract energy from the environment. The respiratory cascade of bacteria culminates in the terminal transfer of electrons onto higher redox potential acceptors in the extracellular space. This general and inducible mechanism of electron efflux during normal bacterial proliferation leads to a characteristic fall in bulk redox potential (Eh), the degree of which is dependent on growth phase, the microbial taxa, and their physiology. Here, we show that the general reducing power of bacteria can be subverted to induce the abiotic production of a carbon-centered radical species for targeted bioorthogonal molecular synthesis. Using two species, Escherichia coli and Salmonella enterica serovar Typhimurium as model microbes, a common redox active aryldiazonium salt is employed to intervene in the terminal respiratory electron flow, affording radical production that is mediated by native redox-active molecular shuttles and active bacterial metabolism. The aryl radicals are harnessed to initiate and sustain a bioorthogonal controlled radical polymerization via reversible addition-fragmentation chain transfer (BacRAFT), yielding a synthetic extracellular matrix of "living" vinyl polymers with predetermined molecular weight and low dispersity. The ability to interface the ubiquitous reducing power of bacteria into synthetic materials design offers a new means for creating engineered living materials with promising adaptive and self-regenerative capabilities.

Flexible Usage and Interconnectivity of Diverse Cell Death Pathways Protect against Intracellular Infection.

Programmed cell death contributes to host defense against pathogens. To investigate the relative importance of pyroptosis, necroptosis, and apoptosis during Salmonella infection, we infected mice and macrophages deficient for diverse combinations of caspases-1, -11, -12, and -8 and receptor interacting serine/threonine kinase 3 (RIPK3). Loss of pyroptosis, caspase-8-driven apoptosis, or necroptosis had minor impact on Salmonella control. However, combined deficiency of these cell death pathways caused loss of bacterial control in mice and their macrophages, demonstrating that host defense can employ varying components of several cell death pathways to limit intracellular infections. This flexible use of distinct cell death pathways involved extensive cross-talk between initiators and effectors of pyroptosis and apoptosis, where initiator caspases-1 and -8 also functioned as executioners when all known effectors of cell death were absent. These findings uncover a highly coordinated and flexible cell death system with in-built fail-safe processes that protect the host from intracellular infections.

Comprehensive analysis of IncC plasmid conjugation identifies a crucial role for the transcriptional regulator AcaB.

The IncC family of broad-host-range plasmids enables the spread of antibiotic resistance genes among human enteric pathogens1-3. Although aspects of IncC plasmid conjugation have been well studied4-9, many roles of conjugation genes have been assigned based solely on sequence similarity. We applied hypersaturated transposon mutagenesis and transposon-directed insertion-site sequencing to determine the set of genes required for IncC conjugation. We identified 27 conjugation genes, comprising 19 that were previously identified (including two regulatory genes, acaDC) and eight not previously associated with conjugation. We show that one previously unknown gene, acaB, encodes a transcriptional regulator that has a crucial role in the regulation of IncC conjugation. AcaB binds upstream of the acaDC promoter to increase acaDC transcription; in turn, AcaDC activates the transcription of IncC conjugation genes. We solved the crystal structure of AcaB at 2.9-Å resolution and used this to guide functional analyses that reveal how AcaB binds to DNA. This improved understanding of IncC conjugation provides a basis for the development of new approaches to reduce the spread of these multi-drug-resistance plasmids.

Global Trends in Proteome Remodeling of the Outer Membrane Modulate Antimicrobial Permeability in Klebsiella pneumoniae.

In Gram-negative bacteria, the permeability of the outer membrane governs rates of antibiotic uptake and thus the efficacy of antimicrobial treatment. Hydrophilic drugs like ß-lactam antibiotics depend on diffusion through pore-forming outer membrane proteins to reach their intracellular targets. In this study, we investigated the distribution of porin genes in more than 2,700 Klebsiella isolates and found a widespread loss of OmpK35 functionality, particularly in those strains isolated from clinical environments. Using a defined set of outer-membrane-remodeled mutants, the major porin OmpK35 was shown to be largely responsible for ß-lactam permeation. Sequence similarity network analysis characterized the porin protein subfamilies and led to discovery of a new porin family member, OmpK38. Structure-based comparisons of OmpK35, OmpK36, OmpK37, OmpK38, and PhoE showed near-identical pore frameworks but defining differences in the sequence characteristics of the extracellular loops. Antibiotic sensitivity profiles of isogenic Klebsiella pneumoniae strains, each expressing a different porin as its dominant pore, revealed striking differences in the antibiotic permeability characteristics of each channel in a physiological context. Since K. pneumoniae is a nosocomial pathogen with high rates of antimicrobial resistance and concurrent mortality, these experiments elucidate the role of porins in conferring specific drug-resistant phenotypes in a global context, informing future research to combat antimicrobial resistance in K. pneumoniaeIMPORTANCEKlebsiella pneumoniae is a pathogen of humans with high rates of mortality and a recognized global rise in incidence of carbapenem-resistant K. pneumoniae (CRKP). The outer membrane of K. pneumoniae forms a permeability barrier that modulates the ability of antibiotics to reach their intracellular target. OmpK35, OmpK36, OmpK37, OmpK38, PhoE, and OmpK26 are porins in the outer membrane of K. pneumoniae, demonstrated here to have a causative relationship to drug resistance phenotypes in a physiological context. The data highlight that currently trialed combination treatments with a carbapenem and ß-lactamase inhibitors could be effective on porin-deficient K. pneumoniae Together with structural data, the results reveal the role of outer membrane proteome remodeling in antimicrobial resistance of K. pneumoniae and point to the role of extracellular loops, not channel parameters, in drug permeation. This significant finding warrants care in the development of phage therapies for K. pneumoniae infections, given the way porin expression will be modulated to confer phage-resistant-and collateral drug-resistant-phenotypes in K. pneumoniae.

The multifunctional enzyme S-adenosylhomocysteine/methylthioadenosine nucleosidase is a key metabolic enzyme in the virulence of Salmonella enterica var Typhimurium.

Key physiological differences between bacterial and mammalian metabolism provide opportunities for the development of novel antimicrobials. We examined the role of the multifunctional enzyme S-adenosylhomocysteine/Methylthioadenosine (SAH/MTA) nucleosidase (Pfs) in the virulence of S. enterica var Typhimurium (S. Typhimurium) in mice, using a defined Pfs deletion mutant (i.e. Δpfs). Pfs was essential for growth of S. Typhimurium in M9 minimal medium, in tissue cultured cells, and in mice. Studies to resolve which of the three known functions of Pfs were key to murine virulence suggested that downstream production of autoinducer-2, spermidine and methylthioribose were non-essential for Salmonella virulence in a highly sensitive murine model. Mass spectrometry revealed the accumulation of SAH in S. Typhimurium Δpfs and complementation of the Pfs mutant with the specific SAH hydrolase from Legionella pneumophila reduced SAH levels, fully restored growth ex vivo and the virulence of S. Typhimurium Δpfs for mice. The data suggest that Pfs may be a legitimate target for antimicrobial development, and that the key role of Pfs in bacterial virulence may be in reducing the toxic accumulation of SAH which, in turn, suppresses an undefined methyltransferase.