Perspectives of key informants before and after implementing UPSIDES peer support in mental health services: qualitative findings from an international multi-site study.

BACKGROUND: Peer support is an essential part of recovery-oriented care worldwide. Contextual factors have an impact on the implementation of peer support work. However, research has paid little attention to similarities and differences of implementation factors in settings varying by income-level and cultural values. The aim of this study is to assess the factors influencing the implementation of a peer support intervention across study sites in low-, middle- and high-income countries in line with the Consolidation Framework for Implementation Research (CFIR). METHOD: 6 focus groups with a total of 54 key informants with relevant contextual (organisational) knowledge regarding implementation facilitators and barriers were conducted at six study sites Ulm and Hamburg (Germany), Butabika (Uganda), Dar es Salaam (Tanzania), Be'er Sheva (Israel), and Pune (India) before and 1.5 years after the start of UPSIDES peer support. Transcripts were analysed using qualitative content analysis. RESULTS: Across study sites key informants reported benefits of peer support for service users and peer support workers as implementation facilitators. At study sites with lower resources, reduced workload for mental health workers and improved access to mental health services through peer support were perceived as implementation facilitators (CFIR Domain 1: Intervention characteristics). The degree of engagement of mental health workers (CFIR Domain 3: Inner Setting/Domain 4: Individuals involved) varied across study sites and was seen either as a barrier (low engagement) or a facilitator (high engagement). Across study sites, adequate training of peer support workers (CFIR Domain 5: Implementation process) was seen as animplementation facilitator, while COVID-19 as well as low resource availability were reported as implementation barriers (CFIR Domain 2: Outer setting). CONCLUSIONS: This study highlights the importance of considering contextual factors when implementing peer support, including previous experience and perceived benefits. Particular attention should be given to organisational benefits such as workload reduction and the allocation of sufficient resources as key drivers in LMICs. In HICs, the potential of organisational benefits for successful implementation should be further investigated and promoted.

Improved genome editing by an engineered CRISPR-Cas12a.

CRISPR-Cas12a is an RNA-guided, programmable genome editing enzyme found within bacterial adaptive immune pathways. Unlike CRISPR-Cas9, Cas12a uses only a single catalytic site to both cleave target double-stranded DNA (dsDNA) (cis-activity) and indiscriminately degrade single-stranded DNA (ssDNA) (trans-activity). To investigate how the relative potency of cis- versus trans-DNase activity affects Cas12a-mediated genome editing, we first used structure-guided engineering to generate variants of Lachnospiraceae bacterium Cas12a that selectively disrupt trans-activity. The resulting engineered mutant with the biggest differential between cis- and trans-DNase activity in vitro showed minimal genome editing activity in human cells, motivating a second set of experiments using directed evolution to generate additional mutants with robust genome editing activity. Notably, these engineered and evolved mutants had enhanced ability to induce homology-directed repair (HDR) editing by 2-18-fold compared to wild-type Cas12a when using HDR donors containing mismatches with crRNA at the PAM-distal region. Finally, a site-specific reversion mutation produced improved Cas12a (iCas12a) variants with superior genome editing efficiency at genomic sites that are difficult to edit using wild-type Cas12a. This strategy establishes a pipeline for creating improved genome editing tools by combining structural insights with randomization and selection. The available structures of other CRISPR-Cas enzymes will enable this strategy to be applied to improve the efficacy of other genome-editing proteins.

Pathogenic Leptospires Limit Dendritic Cell Activation Through Avoidance of TLR4 and TRIF Signaling.

Leptospira interrogans is a bacterial species responsible for leptospirosis, a neglected worldwide zoonosis. Mice and rats are resistant and can become asymptomatic carriers, whereas humans and some other mammals may develop severe forms of leptospirosis. Uncommon among spirochetes, leptospires contain lipopolysaccharide (LPS) in their outer membrane. LPS is highly immunogenic and forms the basis for a large number of serovars. Vaccination with inactivated leptospires elicits a protective immunity, restricted to serovars with related LPS. This protection that lasts in mice, is not long lasting in humans and requires annual boosts. Leptospires are stealth pathogens that evade the complement system and some pattern recognition receptors from the Toll-like (TLR) and Nod-Like families, therefore limiting antibacterial defense. In macrophages, leptospires totally escape recognition by human TLR4, and escape the TRIF arm of the mouse TLR4 pathway. However, very little is known about the recognition and processing of leptospires by dendritic cells (DCs), although they are crucial cells linking innate and adaptive immunity. Here we tested the activation of primary DCs derived from human monocytes (MO-DCs) and mouse bone marrow (BM-DCs) 24h after stimulation with saprophytic or different pathogenic virulent or avirulent L. interrogans. We measured by flow cytometry the expression of DC-SIGN, a lectin involved in T-cell activation, co-stimulation molecules and MHC-II markers, and pro- and anti-inflammatory cytokines by ELISA. We found that exposure to leptospires, live or heat-killed, activated dendritic cells. However, pathogenic L. interrogans, especially from the Icterohaemorraghiae Verdun strain, triggered less marker upregulation and less cytokine production than the saprophytic Leptospira biflexa. In addition, we showed a better activation with avirulent leptospires, when compared to the virulent parental strains in murine BM-DCs. We did not observe this difference in human MO-DCs, suggesting a role for TLR4 in DC stimulation. Accordingly, using BM-DCs from transgenic deficient mice, we showed that virulent Icterohaemorraghiae and Manilae serovars dampened DC activation, at least partly, through the TLR4 and TRIF pathways. This work shows a novel bacterial immune evasion mechanism to limit DC activation and further illustrates the role of the leptospiral LPS as a virulence factor.

Role of the major determinant of polar flagellation FlhG in the endoflagella-containing spirochete Leptospira.

Spirochetes can be distinguished from other bacteria by their spiral-shaped morphology and subpolar periplasmic flagella. This study focused on FlhF and FlhG, which control the spatial and numerical regulation of flagella in many exoflagellated bacteria, in the spirochete Leptospira. In contrast to flhF which seems to be essential in Leptospira, we demonstrated that flhG- mutants in both the saprophyte L. biflexa and the pathogen L. interrogans were less motile than the wild-type strains in gel-like environments but not hyperflagellated as reported previously in other bacteria. Cryo-electron tomography revealed that the distance between the flagellar basal body and the tip of the cell decreased significantly in the flhG- mutant in comparison to wild-type and complemented strains. Additionally, comparative transcriptome analyses of L. biflexa flhG- and wild-type strains showed that FlhG acts as a negative regulator of transcription of some flagellar genes. We found that the L. interrogans flhG- mutant was attenuated for virulence in the hamster model. Cross-species complementation also showed that flhG is not interchangeable between species. Our results indicate that FlhF and FlhG in Leptospira contribute to governing cell motility but our data support the hypothesis that FlhF and FlhG function differently in each bacterial species, including among spirochetes.

Identification of in vivo expressed proteins in live attenuated lipopolysaccharide mutant that mediates heterologous protection against Leptospira spp.

Leptospirosis vaccines that elicit broad protection against a range of pathogenic Leptospira spp. would overcome a major drawback of currently licensed bacterin vaccines. Live attenuated vaccine produced from a lipopolysaccharide (LPS) mutant strain of L. interrogans serovar Manilae M1352 (Live M1352) stimulated better protective efficacy than heat killed M1352 (HK M1352) against a heterologous challenge with L. interrogans serovar Pomona. To identify antigens of Live M1352 potentially responsible for cross protection, in vivo-induced antigen technology (IVIAT), a powerful tool to identify in vivo-induced (ivi) genes expressed during infection, was employed in this study. Pooled sera from hamsters immunized with Live M1352 were sequentially adsorbed with various preparations of in vitro grown M1352. The pre-adsorbed sera were used to screen a genomic expression library of M1352. Nineteen strongly reactive clones were selected for DNA sequencing. These ivi genes are conserved in most Leptospira strains. Four selected genes including LIMLP_04965 (tolB), LIMLP_01535, LIMLP_06785 (fliI), and LIMLP_14930 were confirmed for their upregulated expression in kidneys of infected hamsters by RT-qPCR, suggesting their role in leptospiral infection. These ivi proteins represent potential targets for vaccine candidates that warrant further investigation for their protective efficacy.

Anti-Leptospira immunoglobulin profiling in mice reveals strain specific IgG and persistent IgM responses associated with virulence and renal colonization.

Leptospira interrogans is a pathogenic spirochete responsible for leptospirosis, a neglected, zoonotic reemerging disease. Humans are sensitive hosts and may develop severe disease. Some animal species, such as rats and mice can become asymptomatic renal carriers. More than 350 leptospiral serovars have been identified, classified on the basis of the antibody response directed against the lipopolysaccharide (LPS). Similarly to whole inactivated bacteria used as human vaccines, this response is believed to confer only short-term, serogroup-specific protection. The immune response of hosts against leptospires has not been thoroughly studied, which complicates the testing of vaccine candidates. In this work, we studied the immunoglobulin (Ig) profiles in mice infected with L. interrogans over time to determine whether this humoral response confers long-term protection after homologous challenge six months post-infection. Groups of mice were injected intraperitoneally with 2×107 leptospires of one of three pathogenic serovars (Manilae, Copenhageni or Icterohaemorrhagiae), attenuated mutants or heat-killed bacteria. Leptospira-specific immunoglobulin (IgA, IgM, IgG and 4 subclasses) produced in the first weeks up to 6 months post-infection were measured by ELISA. Strikingly, we found sustained high levels of IgM in mice infected with the pathogenic Manilae and Copenhageni strains, both colonizing the kidney. In contrast, the Icterohaemorrhagiae strain did not lead to kidney colonization, even at high dose, and triggered a classical IgM response that peaked at day 8 post-infection and disappeared. The virulent Manilae and Copenhageni serovars elicited high levels and similar profiles of IgG subclasses in contrast to Icterohaemorrhagiae strains that stimulated weaker antibody responses. Inactivated heat-killed Manilae strains elicited very low responses. However, all mice pre-injected with leptospires challenged with high doses of homologous bacteria did not develop acute leptospirosis, and all antibody responses were boosted after challenge. Furthermore, we showed that 2 months post-challenge, mice pre-infected with the attenuated M895 Manilae LPS mutant or heat-killed bacterin were completely protected against renal colonization. In conclusion, we observed a sustained IgM response potentially associated with chronic leptospiral renal infection. We also demonstrated in mice different profiles of protective and cross-reactive antibodies after L. interrogans infection, depending on the serovar and virulence of strains.

Correction: Leptospiral LPS escapes mouse TLR4 internalization and TRIF-associated antimicrobial responses through O antigen and associated lipoproteins.

[This corrects the article DOI: 10.1371/journal.ppat.1008639.].

Leptospiral LPS escapes mouse TLR4 internalization and TRIF­associated antimicrobial responses through O antigen and associated lipoproteins.

Leptospirosis is a worldwide re-emerging zoonosis caused by pathogenic Leptospira spp. All vertebrate species can be infected; humans are sensitive hosts whereas other species, such as rodents, may become long-term renal carrier reservoirs. Upon infection, innate immune responses are initiated by recognition of Microbial Associated Molecular Patterns (MAMPs) by Pattern Recognition Receptors (PRRs). Among MAMPs, the lipopolysaccharide (LPS) is recognized by the Toll-Like-Receptor 4 (TLR4) and activates both the MyD88-dependent pathway at the plasma membrane and the TRIF-dependent pathway after TLR4 internalization. We previously showed that leptospiral LPS is not recognized by the human-TLR4, whereas it signals through mouse-TLR4 (mTLR4), which mediates mouse resistance to acute leptospirosis. However, although resistant, mice are known to be chronically infected by leptospires. Interestingly, the leptospiral LPS has low endotoxicity in mouse cells and is an agonist of TLR2, the sensor for bacterial lipoproteins. Here, we investigated the signaling properties of the leptospiral LPS in mouse macrophages. Using confocal microscopy and flow cytometry, we showed that the LPS of L. interrogans did not induce internalization of mTLR4, unlike the LPS of Escherichia coli. Consequently, the LPS failed to induce the production of the TRIF-dependent nitric oxide and RANTES, both important antimicrobial responses. Using shorter LPS and LPS devoid of TLR2 activity, we further found this mTLR4-TRIF escape to be dependent on both the co-purifying lipoproteins and the full-length O antigen. Furthermore, our data suggest that the O antigen could alter the binding of the leptospiral LPS to the co-receptor CD14 that is essential for TLR4-TRIF activation. Overall, we describe here a novel leptospiral immune escape mechanism from mouse macrophages and hypothesize that the LPS altered signaling could contribute to the stealthiness and chronicity of the leptospires in mice.

Disruption of the Burkholderia pseudomallei two-component signal transduction system BbeR-BbeS leads to increased extracellular DNA secretion and altered biofilm formation.

Two-component signal transduction systems (TCSTS) are abundant among prokaryotes and regulate important functions, including drug resistance and virulence. The Gram-negative bacterium Burkholderia pseudomallei, which causes the severe infectious disease melioidosis, encodes 136 putative TCSTS components. In silico analyses of these TCSTS indicated that the predicted BbeR-BbeS system (BPSL1036-BPSL1037) displayed significant amino acid sequence similarity to the Shigella flexneri virulence-associated OmpR-EnvZ osmoregulator. To assess the function of the B. pseudomallei BbeR-BbeS system, we constructed by allelic exchange a ΔbbeRS double mutant strain lacking both genes, and single ΔbbeR and ΔbbeS mutants. All three mutant strains caused disease in the BALB/c acute melioidosis model at the same rate as the wild-type strain, displayed unchanged swarming motility on semi-solid medium, and were unaffected for viability on high-osmolarity media. However, when cultured at 37 °C for at least 14 days, ΔbbeS and ΔbbeR colonies developed a distinct, hypermucoid morphology absent in similarly-cultured wild-type colonies. At both 30 °C and 37 °C, these hypermucoid strains produced wild-type levels of type I capsule but released increased quantities of extracellular DNA (eDNA). Upon static growth in liquid medium, all B. pseudomallei strains produced pellicle biofilms that contained DNA in close association with bacterial cells; however, the ΔbbeS and ΔbbeR strains produced increased biofilms with altered microscopic architecture compared to the wild-type. Unusually, while the ΔbbeS and ΔbbeR single-deletion mutants displayed clear phenotypes, the ΔbbeRS double-deletion mutant was indistinguishable from the wild-type strain. We propose that BbeR-BbeS indirectly affects eDNA secretion and biofilm formation through cross-talk with one or more other TCSTS.

Heterologous protection elicited by a live, attenuated, Leptospira vaccine.

A previously-described live, attenuated vaccine (M1352, serovar Manilae, serogroup Pyrogenes) was tested in the hamster model of infection for cross-protective immunity. The vaccine elicited strong, significant cross-protection against lethal infection by strains representing four serologically distinct leptospiral serovars (Grippotyphosa, Australis, Canicola, and Autumnalis). Combined with our previously reported protection against serovars Pomona and Manilae, this work demonstrates unequivocal proof of concept for cross-protective immunity in leptospirosis.

Antimicrobial Resistance in Leptospira, Brucella, and Other Rarely Investigated Veterinary and Zoonotic Pathogens.

Leptospira, Brucella, and Borrelia are major agents of zoonotic disease, causing high morbidity and, in some cases, significant mortality in humans. For all three genera, prompt diagnosis and appropriate antimicrobial therapy are required to prevent the development of chronic, debilitating illness. Leptospira spp. are intrinsically resistant to several antimicrobial classes; however, there is little evidence in the literature for development of acquired resistance to antimicrobial agents used for clinical treatment of acute leptospirosis. For Brucella infections, there are numerous reports of relapses following therapy, but it is unclear whether this is due to sequestration within infected sites (e.g., bone) or the development of acquired resistance. Brucella have maintained their susceptibility to doxycycline and rifampicin, which in combination remain the most common treatments of brucellosis in humans. In vitro induced point mutations are described as imparting resistance to rifampicin (rpoB) and fluoroquinolones (gyrA). The clinical significance of these mutations is unclear. For Borrelia burgdorferi, although acquired resistance to some antimicrobial agents has been described, resistance due to bacterial persister cells surviving in the presence of antimicrobial, with no apparent increase in the MIC of the organism, have been recently described. Of the remaining veterinary fastidious pathogens, Lawsonia intracellularis is the most interesting from an antimicrobial resistance perspective because it can only be grown in cell culture, making in vitro susceptibility testing challenging. MIC testing has been undertaken on a small number of isolates, and some differences in susceptibility to macrolides have been demonstrated between isolates obtained from different regions.

Leptospiral Genomics and Pathogenesis.

Until about 15 years ago, the molecular and cellular basis for pathogenesis in leptospirosis was virtually unknown. The determination of the first full genome sequence in 2003 was followed rapidly by other whole genome sequences, whose availability facilitated the development of transposon mutagenesis and then directed mutagenesis of pathogenic Leptospira spp. The combination of genomics, transcriptomics and mutant construction and characterisation has resulted in major progress in our understanding of the taxonomy and biology of Leptospira. The most recent advances are analysed and discussed in this chapter.

Lvr, a Signaling System That Controls Global Gene Regulation and Virulence in Pathogenic Leptospira.

Leptospirosis is an emerging zoonotic disease with more than 1 million cases annually. Currently there is lack of evidence for signaling pathways involved during the infection process of Leptospira. In our comprehensive genomic analysis of 20 Leptospira spp. we identified seven pathogen-specific Two-Component System (TCS) proteins. Disruption of two these TCS genes in pathogenic Leptospira strain resulted in loss-of-virulence in a hamster model of leptospirosis. Corresponding genes lvrA and lvrB (leptospira virulence regulator) are juxtaposed in an operon and are predicted to encode a hybrid histidine kinase and a hybrid response regulator, respectively. Transcriptome analysis of lvr mutant strains with disruption of one (lvrB) or both genes (lvrA/B) revealed global transcriptional regulation of 850 differentially expressed genes. Phosphotransfer assays demonstrated that LvrA phosphorylates LvrB and predicted further signaling downstream to one or more DNA-binding response regulators, suggesting that it is a branched pathway. Phylogenetic analyses indicated that lvrA and lvrB evolved independently within different ecological lineages in Leptospira via gene duplication. This study uncovers a novel-signaling pathway that regulates virulence in pathogenic Leptospira (Lvr), providing a framework to understand the molecular bases of regulation in this life-threatening bacterium.

Characterization of Two Novel Lipopolysaccharide Phosphoethanolamine Transferases in Pasteurella multocida and Their Role in Resistance to Cathelicidin-2.

The lipopolysaccharide (LPS) produced by the Gram-negative bacterial pathogen Pasteurella multocida has phosphoethanolamine (PEtn) residues attached to lipid A, 3-deoxy-d-manno-octulosonic acid (Kdo), heptose, and galactose. In this report, we show that PEtn is transferred to lipid A by the P. multocida EptA homologue, PetL, and is transferred to galactose by a novel PEtn transferase that is unique to P. multocida called PetG. Transcriptomic analyses indicated that petL expression was positively regulated by the global regulator Fis and negatively regulated by an Hfq-dependent small RNA. Importantly, we have identified a novel PEtn transferase called PetK that is responsible for PEtn addition to the single Kdo molecule (Kdo1), directly linked to lipid A in the P. multocida glycoform A LPS. In vitro assays showed that the presence of a functional petL and petK, and therefore the presence of PEtn on lipid A and Kdo1, was essential for resistance to the cationic, antimicrobial peptide cathelicidin-2. The importance of PEtn on Kdo1 and the identification of the transferase responsible for this addition have not previously been shown. Phylogenetic analysis revealed that PetK is the first representative of a new family of predicted PEtn transferases. The PetK family consists of uncharacterized proteins from a range of Gram-negative bacteria that produce LPS glycoforms with only one Kdo molecule, including pathogenic species within the genera Vibrio, Bordetella, and Haemophilus We predict that many of these bacteria will require the addition of PEtn to Kdo for maximum protection against host antimicrobial peptides.

Adipose tissue is the first colonization site of Leptospira interrogans in subcutaneously infected hamsters.

Leptospirosis is one of the most widespread zoonoses in the world, and its most severe form in humans, "Weil's disease," may lead to jaundice, hemorrhage, renal failure, pulmonary hemorrhage syndrome, and sometimes,fatal multiple organ failure. Although the mechanisms underlying jaundice in leptospirosis have been gradually unraveled, the pathophysiology and distribution of leptospires during the early stage of infection are not well understood. Therefore, we investigated the hamster leptospirosis model, which is the accepted animal model of human Weil's disease, by using an in vivo imaging system to observe the whole bodies of animals infected with Leptospira interrogans and to identify the colonization and growth sites of the leptospires during the early phase of infection. Hamsters, infected subcutaneously with 104 bioluminescent leptospires, were analyzed by in vivo imaging, organ culture, and microscopy. The results showed that the luminescence from the leptospires spread through each hamster's body sequentially. The luminescence was first detected at the injection site only, and finally spread to the central abdomen, in the liver area. Additionally, the luminescence observed in the adipose tissue was the earliest detectable compared with the other organs, indicating that the leptospires colonized the adipose tissue at the early stage of leptospirosis. Adipose tissue cultures of the leptospires became positive earlier than the blood cultures. Microscopic analysis revealed that the leptospires colonized the inner walls of the blood vessels in the adipose tissue. In conclusion, this is the first study to report that adipose tissue is an important colonization site for leptospires, as demonstrated by microscopy and culture analyses of adipose tissue in the hamster model of Weil's disease.

Perturbation of the two-component signal transduction system, BprRS, results in attenuated virulence and motility defects in Burkholderia pseudomallei.

BACKGROUND: Burkholderia pseudomallei is the causative agent of melioidosis, a severe invasive disease of humans and animals. Initial screening of a B. pseudomallei signature-tagged mutagenesis library identified an attenuated mutant with a transposon insertion in a gene encoding the sensor component of an uncharacterised two-component signal transduction system (TCSTS), which we designated BprRS. RESULTS: Single gene inactivation of either the response regulator gene (bprR) or the sensor histidine kinase gene (bprS) resulted in mutants with reduced swarming motility and reduced virulence in mice. However, a bprRS double mutant was not attenuated for virulence and displayed wild-type levels of motility. The transcriptomes of the bprS, bprR and bprRS mutants were compared with the transcriptome of the parent strain K96243. Inactivation of the entire BprRS TCSTS (bprRS double mutant) resulted in altered expression of only nine genes, including both bprR and bprS, five phage-related genes and bpss0686, encoding a putative 5, 10-methylene tetrahydromethanopterin reductase involved in one carbon metabolism. In contrast, the transcriptomes of each of the bprR and bprS single gene mutants revealed more than 70 differentially expressed genes common to both mutants, including regulatory genes and those required for flagella assembly and for the biosynthesis of the cytotoxic polyketide, malleilactone. CONCLUSIONS: Inactivation of the entire BprRS TCSTS did not alter virulence or motility and very few genes were differentially expressed indicating that the definitive BprRS regulon is relatively small. However, loss of a single component, either the sensor histidine kinase BprS or its cognate response regulator BprR, resulted in significant transcriptomic and phenotypic differences from the wild-type strain. We hypothesize that the dramatically altered phenotypes of these single mutants are the result of cross-regulation with one or more other TCSTSs and concomitant dysregulation of other key regulatory genes.

What Makes a Bacterial Species Pathogenic?:Comparative Genomic Analysis of the Genus Leptospira.

Leptospirosis, caused by spirochetes of the genus Leptospira, is a globally widespread, neglected and emerging zoonotic disease. While whole genome analysis of individual pathogenic, intermediately pathogenic and saprophytic Leptospira species has been reported, comprehensive cross-species genomic comparison of all known species of infectious and non-infectious Leptospira, with the goal of identifying genes related to pathogenesis and mammalian host adaptation, remains a key gap in the field. Infectious Leptospira, comprised of pathogenic and intermediately pathogenic Leptospira, evolutionarily diverged from non-infectious, saprophytic Leptospira, as demonstrated by the following computational biology analyses: 1) the definitive taxonomy and evolutionary relatedness among all known Leptospira species; 2) genomically-predicted metabolic reconstructions that indicate novel adaptation of infectious Leptospira to mammals, including sialic acid biosynthesis, pathogen-specific porphyrin metabolism and the first-time demonstration of cobalamin (B12) autotrophy as a bacterial virulence factor; 3) CRISPR/Cas systems demonstrated only to be present in pathogenic Leptospira, suggesting a potential mechanism for this clade's refractoriness to gene targeting; 4) finding Leptospira pathogen-specific specialized protein secretion systems; 5) novel virulence-related genes/gene families such as the Virulence Modifying (VM) (PF07598 paralogs) proteins and pathogen-specific adhesins; 6) discovery of novel, pathogen-specific protein modification and secretion mechanisms including unique lipoprotein signal peptide motifs, Sec-independent twin arginine protein secretion motifs, and the absence of certain canonical signal recognition particle proteins from all Leptospira; and 7) and demonstration of infectious Leptospira-specific signal-responsive gene expression, motility and chemotaxis systems. By identifying large scale changes in infectious (pathogenic and intermediately pathogenic) vs. non-infectious Leptospira, this work provides new insights into the evolution of a genus of bacterial pathogens. This work will be a comprehensive roadmap for understanding leptospirosis pathogenesis. More generally, it provides new insights into mechanisms by which bacterial pathogens adapt to mammalian hosts.

The RNA-Binding Chaperone Hfq Is an Important Global Regulator of Gene Expression in Pasteurella multocida and Plays a Crucial Role in Production of a Number of Virulence Factors, Including Hyaluronic Acid Capsule.

The Gram-negative bacterium Pasteurella multocida is the causative agent of a number of economically important animal diseases, including avian fowl cholera. Numerous P. multocida virulence factors have been identified, including capsule, lipopolysaccharide (LPS), and filamentous hemagglutinin, but little is known about how the expression of these virulence factors is regulated. Hfq is an RNA-binding protein that facilitates riboregulation via interaction with small noncoding RNA (sRNA) molecules and their mRNA targets. Here, we show that a P. multocida hfq mutant produces significantly less hyaluronic acid capsule during all growth phases and displays reduced in vivo fitness. Transcriptional and proteomic analyses of the hfq mutant during mid-exponential-phase growth revealed altered transcript levels for 128 genes and altered protein levels for 78 proteins. Further proteomic analyses of the hfq mutant during the early exponential growth phase identified 106 proteins that were produced at altered levels. Both the transcript and protein levels for genes/proteins involved in capsule biosynthesis were reduced in the hfq mutant, as were the levels of the filamentous hemagglutinin protein PfhB2 and its secretion partner LspB2. In contrast, there were increased expression levels of three LPS biosynthesis genes, encoding proteins involved in phosphocholine and phosphoethanolamine addition to LPS, suggesting that these genes are negatively regulated by Hfq-dependent mechanisms. Taken together, these data provide the first evidence that Hfq plays a crucial role in regulating the global expression of P. multocida genes, including the regulation of key P. multocida virulence factors, capsule, LPS, and filamentous hemagglutinin.

Protective efficacy afforded by live Pasteurella multocida vaccines in chickens is independent of lipopolysaccharide outer core structure.

Pasteurella multocida is a major animal pathogen that causes a range of diseases including fowl cholera. P. multocida infections result in considerable losses to layer and breeder flocks in poultry industries worldwide. Both killed whole-cell and live-attenuated vaccines are available; these vaccines vary in their protective efficacy, particularly against heterologous strains. Moreover, until recently there was no knowledge of P. multocida LPS genetics and structure to determine precisely how LPS structure affects the protective capacity of these vaccines. In this study we show that defined lipopolysaccharide (LPS) mutants presented as killed whole-cell vaccines elicited solid protective immunity only against P. multocida challenge strains expressing highly similar or identical LPS structures. This finding indicates that vaccination of commercial flocks with P. multocida killed cell formulations will not protect against strains producing an LPS structure different to that produced by strains included in the vaccine formulation. Conversely, protective immunity conferred by vaccination with live P. multocida strains was found to be largely independent of LPS structure. Birds vaccinated with a range of live mutants belonging to the L1 and L3 LPS genotypes, each expressing a specific truncated LPS structure, were protected against challenge with the parent strain. Moreover, birds vaccinated with any of the five LPS mutants belonging to the L1 LPS genotype were also protected against challenge with an unrelated strain and two of the five groups vaccinated with live LPS mutants belonging to the L3 genotype were protected against challenge with an unrelated strain. In summary, vaccination with live P. multocida aroA mutants producing full-length L1 or L3 LPS or vaccination with live strains producing shortened L1 LPS elicited strong protective immunity against both homologous and heterologous challenge.

The Burkholderia pseudomallei Proteins BapA and BapC Are Secreted TTSS3 Effectors and BapB Levels Modulate Expression of BopE.

Many Gram-negative pathogens use a type III secretion system (TTSS) for the injection of bacterial effector proteins into host cells. The injected effector proteins play direct roles in modulation of host cell pathways for bacterial benefit. Burkholderia pseudomallei, the causative agent of melioidosis, expresses three different TTSSs. One of these systems, the TTSS3, is essential for escape from host endosomes and therefore intracellular survival and replication. Here we have characterized three putative TTSS3 proteins; namely BapA, BapB and BapC. By employing a tetracysteine (TC)-FlAsH™ labelling technique to monitor the secretion of TC-tagged fusion proteins, BapA and BapC were shown to be secreted during in vitro growth in a TTSS3-dependant manner, suggesting a role as TTSS3 effectors. Furthermore, we constructed B. pseudomallei bapA, bapB and bapC mutants and used the well-characterized TTSS3 effector BopE as a marker of secretion to show that BapA, BapB and BapC are not essential for the secretion process. However, BopE transcription and secretion were significantly increased in the bapB mutant, suggesting that BapB levels modulate BopE expression. In a BALB/c mouse model of acute melioidosis, the bapA, bapB and bapC mutants showed a minor reduction of in vivo fitness. Thus, this study defines BapA and BapC as novel TTSS3 effectors, BapB as a regulator of BopE production, and all three as necessary for full B. pseudomallei in vivo fitness.