Recombinant expression of Yersinia ruckeri outer membrane proteins in Escherichia coli extracellular vesicles.

The secretion of extracellular vesicles (EVs) is a common process in Gram-negative bacteria and can be exploited for biotechnological applications. EVs pose a self-adjuvanting, non-replicative vaccine platform, where membrane and antigens are presented to the host immune system in a non-infectious fashion. The secreted quantity of EVs varies between Gram-negative bacterial species and is comparatively high in the model bacterium E. coli. The outer membrane proteins OmpA and OmpF of the fish pathogen Y. ruckeri have been proposed as vaccine candidates to prevent enteric redmouth disease in aquaculture. In this work, Y.ruckeri OmpA or OmpF were expressed in E. coli and recombinant EVs were isolated. To avoid competition between endogenous E. coli OmpA or OmpF, Y. ruckeri OmpA and OmpF were expressed in E. coli strains lacking ompA, ompF, and in a quadruple knockout strain where the four major outer membrane protein genes ompA, ompC, ompF and lamB were removed. Y.ruckeri OmpA and OmpF were successfully expressed in EVs derived from the E. coli mutants as verified by SDS-PAGE, heat modifiability and proteomic analysis using mass-spectrometry. Transmission electron microscopy revealed the presence of EVs in all E. coli strains, and increased EV concentrations were detected when expressing Y. ruckeri OmpA or OmpF in recombinant EVs compared to empty vector controls as verified by nanoparticle tracking analysis. These results show that E. coli can be utilized as a vector for production of EVs expressing outer membrane antigens from Y. ruckeri.

Outer membrane vesicles from Piscirickettsia salmonis induce the expression of inflammatory genes and production of IgM in Atlantic salmon Salmo salar.

Piscirickettsiosis outbreaks due to Piscirickettsia salmonis occur globally in the Chilean salmon aquaculture generating significant monetary losses in the industry. P. salmonis secretes outer membrane vesicles (OMVs) which are naturally non-replicating and highly immunogenic spherical nanoparticles. P. salmonis OMVs has been shown to induce immune response in zebrafish; however, the immune response induced by these vesicles in salmonids has not been evaluated. In this study, we inoculated Atlantic salmon with 10 and 30 µg doses of P. salmonis OMVs and took samples for 12 days. qPCR analysis indicated an inflammatory response. Thus, the inflammatory genes evaluated were up- or down-regulated at several times in liver, head kidney and spleen. In addition, the liver was the organ most immune-induced, mainly in the 30 µg-dose. Interestingly, co-expression of pro- and anti-inflammatory cytokines was evidenced by the prominent expression of il-10 at day 1 in spleen and also in head kidney on days 3, 6 and 12, while il-10 and tgf-ß were up-regulated on days 3, 6 and 12 in liver. Importantly, we detected the production of IgM against proteins of P. salmonis in the serum collected from immunized fish after 14 days. Thus, 40 and 400 µg OMVs induced the production of highest IgM levels; however, no statistical difference in the immunoglobulin levels produced by these OMVs doses were detected. The current study provides evidence that OMVs released by P. salmonis induced a pro-inflammatory responses and IgM production in S. salar, while regulatory genes were induced in order to regulate their effects and achieve the balance of the inflammatory response.

Expanding Francisella models: Pairing up the soil amoeba Dictyostelium with aquatic Francisella.

The bacterial genus Francisella comprises highly pathogenic species that infect mammals, arthropods, fish and protists. Understanding virulence and host defense mechanisms of Francisella infection relies on multiple animal and cellular model systems. In this review, we want to summarize the most commonly used Francisella host model platforms and highlight novel, alternative model systems using aquatic Francisella species. Established mouse and macrophage models contributed extensively to our understanding of Francisella infection. However, murine and human cells display significant differences in their response to Francisella infection. The zebrafish and the amoeba Dictyostelium are well-established model systems for host-pathogen interactions and open up opportunities to investigate bacterial virulence and host defense. Comparisons between model systems using human and fish pathogenic Francisella species revealed shared virulence strategies and pathology between them. Hence, zebrafish and Dictyostelium might complement current model systems to find new vaccine candidates and contribute to our understanding of Francisella infection.

Immunomodulatory properties of Concholepas concholepas hemocyanin against francisellosis in a zebrafish model.

The development of vaccines for aquaculture has been an important milestone in providing a continuous and sustainable production. Most of the vaccines currently on the market for aquaculture include oil as adjuvant. Nevertheless, several studies reported an occurrence of side effects after their use in farmed fish. As a result, there is a need for new and improved adjuvants that can stimulate the immune system while causing as few side-effects as possible. Hemocyanins are versatile macromolecules with strong immunogenic and immunomodulatory properties. Due to these characteristics, hemocyanin from Concholepas concholepas (CCH) has been biochemically characterized and evaluated as vaccine adjuvant in mice and humans. Francisellosis is a chronic granulomatous disease, which can result in high mortality depending on the host. The disease is caused by the facultative intracellular Gram-negative bacteria Francisella noatunensis, which remains an unsolved problem for the aquaculture, as no efficient vaccines are available. The aim of the present work was to investigate the immunoregulatory properties of CCH against francisellosis in an experimental zebrafish model. When immunized with CCH, zebrafish were protected from subsequent challenge with a lethal dose of Francisella noatunensis subsp. orientalis. Subsequently the mRNA expression levels of several immune-related genes were studied, including mhcii, il12a, tnfα and ifng1-1. Taken together, the data report the immunoregulatory properties of CCH and its potential use as a vaccine adjuvant for aquaculture.

Membrane vesicles from Piscirickettsia salmonis induce protective immunity and reduce development of salmonid rickettsial septicemia in an adult zebrafish model.

Infections caused by the facultative intracellular bacterial pathogen Piscirickettsia salmonis remains an unsolved problem for the aquaculture as no efficient treatments have been developed. As a result, substantial amounts of antibiotic have been used to limit salmonid rickettsial septicemia (SRS) disease outbreaks. The antibiotic usage has not reduced the occurrence, but lead to an increase in resistant strains, underlining the need for new treatment strategies. P. salmonis produce membrane vesicles (MVs); small spherical structures know to contain a variety of bacterial components, including proteins, lipopolysaccharides (LPS), DNA and RNA. MVs mimics' in many aspects their mother cell, and has been reported as alternative vaccine candidates. Here, MVs from P. salmonis was isolated and evaluated as a vaccine candidate against SRS in an adult zebrafish infection model. When zebrafish was immunized with MVs they were protected from subsequent challenge with a lethal dose of P. salmonis. Histological analysis showed a reduced bacterial load upon challenge in the MV immunized group, and the mRNA expression levels of several immune related genes altered, including mpeg1.1, tnfα, il1b, il10 and il6. The MVs induced the secretion of IgM upon immunization, indicating an immunogenic effect of the vesicles. Taken together, the data demonstrate a vaccine potential of MVs against P. salmonis.

Francisella noatunensis ssp. noatunensis iglC deletion mutant protects adult zebrafish challenged with acute mortality dose of wild-type strain.

The intracellular fish pathogen Francisella noatunensis remains an unsolved problem for aquaculture worldwide and an efficient vaccine is needed. In Francisella sp., IglC is an important virulence factor necessary for intracellular growth and escape from phagolysosomes. Deletion of the intracellular growth locus C (iglC) in Francisella sp. causes attenuation, but vaccine potential has only been attributed to ΔiglC from Francisella noatunensis ssp. orientalis, a warm-water fish pathogen. A ΔiglC mutant was constructed in the cold-water fish pathogen F. noatunensis ssp. noatunensis (Fnn), which causes francisellosis in Atlantic cod; the mutant was assessed in primary head kidney leucocytes from Atlantic cod. Fluorescence microscopy revealed reduced growth, while qPCR revealed an initial increase followed by a reduction in mutant genomes. Mutant-infected cod leucocytes presented higher interleukin 1 beta (il1ß) and interleukin 8 (il8) transcription than wild-type (WT)-infected cells. Two doses of mutant and WT were tested in an adult zebrafish model whereupon 3 × 109 CFU caused acute disease and 3 × 107 CFU caused low mortality regardless of strain. However, splenomegaly developed only in the WT-infected zebrafish. Immunization with 7 × 106 CFU of Fnn ΔiglC protected zebrafish against challenge with a lethal dose of Fnn WT, and bacterial load was minimized within 28 d. Immunized fish had lower interleukin 6 (il6) and il8 transcription in kidney and prolonged interferon-gamma (ifng) transcription in spleens after challenge compared with non-immunized fish. Our data suggest an immunogenic potential of Fnn ΔiglC and indicate important cytokines associated with francisellosis pathogenesis and protection.

Treatment of Francisella infections via PLGA- and lipid-based nanoparticle delivery of antibiotics in a zebrafish model.

We tested the efficiency of 2 different antibiotics, rifampicin and oxolinic acid, against an established infection caused by fish pathogen Francisella noatunensis ssp. orientalis (F.n.o.) in zebrafish. The drugs were tested in the free form as well as encapsulated into biodegradable nanoparticles, either polylactic-co-glycolic acid (PLGA) nanoparticles or nanostructured lipid carriers. The most promising therapies were PLGA-rifampicin nanoparticles and free oxolinic acid; the PLGA nanoparticles significantly delayed embryo mortality while free oxolinic acid prevented it. Encapsulation of rifampicin in both PLGA and nanostructured lipid carriers enhanced its efficiency against F.n.o. infection relative to the free drug. We propose that the zebrafish model is a robust, rapid system for initial testing of different treatments of bacterial diseases important for aquaculture.

Dissection of Francisella-Host Cell Interactions in Dictyostelium discoideum.

Francisella bacteria cause severe disease in both vertebrates and invertebrates and include one of the most infectious human pathogens. Mammalian cell lines have mainly been used to study the mechanisms by which Francisella manipulates its host to replicate within a large variety of hosts and cell types, including macrophages. Here, we describe the establishment of a genetically and biochemically tractable infection model: the amoeba Dictyostelium discoideum combined with the fish pathogen Francisella noatunensis subsp. noatunensis. Phagocytosed F. noatunensis subsp. noatunensis interacts with the endosomal pathway and escapes further phagosomal maturation by translocating into the host cell cytosol. F. noatunensis subsp. noatunensis lacking IglC, a known virulence determinant required for Francisella intracellular replication, follows the normal phagosomal maturation and does not grow in Dictyostelium. The attenuation of the F. noatunensis subsp. noatunensis ΔiglC mutant was confirmed in a zebrafish embryo model, where growth of F. noatunensis subsp. noatunensis ΔiglC was restricted. In Dictyostelium, F. noatunensis subsp. noatunensis interacts with the autophagic machinery. The intracellular bacteria colocalize with autophagic markers, and when autophagy is impaired (Dictyostelium Δatg1), F. noatunensis subsp. noatunensis accumulates within Dictyostelium cells. Altogether, the Dictyostelium-F. noatunensis subsp. noatunensis infection model recapitulates the course of infection described in other host systems. The genetic and biochemical tractability of the system allows new approaches to elucidate the dynamic interactions between pathogenic Francisella and its host organism.

Vaccination with outer membrane vesicles from Francisella noatunensis reduces development of francisellosis in a zebrafish model.

Infection of fish with the facultative intracellular bacterium Francisella noatunensis remains an unresolved problem for aquaculture industry worldwide as it is difficult to vaccinate against without using live attenuated vaccines. Outer membrane vesicles (OMVs) are biological structures shed by Gram-negative bacteria in response to various environmental stimuli. OMVs have successfully been used to vaccinate against both intracellular and extracellular pathogens, due to an ability to stimulate innate, cell-mediated and humoral immune responses. We show by using atomic force and electron microscopy that the fish pathogenic bacterium F. noatunensis subspecies noatunensis (F.n.n.) shed OMVs both in vitro into culture medium and in vivo in a zebrafish infection model. The main protein constituents of the OMV are IglC, PdpD and PdpA, all known Francisella virulence factors, in addition to the outer membrane protein FopA and the chaperonin GroEL, as analyzed by mass spectrometry. The vesicles, when used as a vaccine, reduced proliferation of the bacterium and protected zebrafish when subsequently challenged with a high dose of F.n.n. without causing adverse effects for the host. Also granulomatous responses were reduced in F.n.n.-challenged zebrafish after OMV vaccination. Taken together, the data support the possible use of OMVs as vaccines against francisellosis in fish.

Establishment of three Francisella infections in zebrafish embryos at different temperatures.

Francisella spp. are facultative intracellular pathogens identified in increasingly diverse hosts, including mammals. F. noatunensis subsp. orientalis and F. noatunensis subsp. noatunensis infect fish inhabiting warm and cold waters, respectively, while F. tularensis subsp. novicida is highly infectious for mice and has been widely used as a model for the human pathogen F. tularensis. Here, we established zebrafish embryo infection models of fluorescently labeled F. noatunensis subsp. noatunensis, F. noatunensis subsp. orientalis, and F. tularensis subsp. novicida at 22, 28, and 32°C, respectively. All infections led to significant bacterial growth, as shown by reverse transcription-quantitative PCR (RT-qPCR), and to a robust proinflammatory immune response, dominated by increased transcription of tumor necrosis factor alpha (TNF-α) and interleukin-1ß (IL-1ß). F. noatunensis subsp. orientalis was the most virulent, F. noatunensis subsp. noatunensis caused chronic infection, and F. tularensis subsp. novicida showed moderate virulence and led to formation of relatively small granuloma-like structures. The use of transgenic zebrafish strains with enhanced green fluorescent protein (EGFP)-labeled immune cells revealed their detailed interactions with Francisella species. All three strains entered preferentially into macrophages, which eventually assembled into granuloma-like structures. Entry into neutrophils was also observed, though the efficiency of this event depended on the route of infection. The results demonstrate the usefulness of the zebrafish embryo model for studying infections caused by different Francisella species at a wide range of temperatures and highlight their interactions with immune cells.

Host specificity and clade dependent distribution of putative virulence genes in Moritella viscosa.

Moritella viscosa is the aetiological agent of winter-ulcer disease in farmed salmonids in the North Atlantic. Previously, two major (typical and variant) genetic clades have been demonstrated within this bacterial species, one of which is almost solely related to disease in Atlantic salmon (Salmo salar). In the present study infection trials demonstrated that 'typical' M. viscosa isolated from Norwegian Atlantic salmon was highly virulent in this fish species but resulted in lower levels of mortality in rainbow trout. 'Variant' M. viscosa isolated from rainbow trout resulted in modest mortality levels in both Atlantic salmon and rainbow trout. To investigate the possible genetic background for inter-strain virulence differences, 38 M. viscosa isolates of diverse geographical origin and host species and a number of other Moritella spp. were investigated for the presence/absence of putative virulence related homologs. All isolates were positive for DNA sequences coding for; the Type VI secretion ATPase (clpV), hemolysin co-regulated protein (hcp), bacterioferritins (bfrA and bfrB), lectin (hemG), phospholipase D (pld), multifunctional autoprocessing repeats-in-toxin (martxA), aerolysin (aer), invasin (inv), and cytotoxic necrotizing factor (cnf), with the exception of one isolate in which cnf could not be confirmed. The product of an ABC transporter metal-binding lipoprotein (mat) was consistently detected although 11 isolates, all phylogenetically related, appear to produce a truncated version. A putative insecticidal toxin complex (mitABC) was detected almost exclusively in 'typical' Atlantic salmon isolates, and our data indicate that this complex of genes is expressed and co-transcribed. Transmission electron microscopy investigation revealed pili and flagella surface structures on nine M. viscosa representing both typical and variant isolates. Our results provide strong support for the existence of host specificity/high virulence in 'typical' M. viscosa related to Atlantic salmon. The gene distribution also provides further support for the genetic division within M. viscosa, and constitutes a basis for further study of the importance of the mitABC complex in winter-ulcer pathogenesis.

Crystal structures of a CTXphi pIII domain unbound and in complex with a Vibrio cholerae TolA domain reveal novel interaction interfaces.

Vibrio cholerae colonize the small intestine where they secrete cholera toxin, an ADP-ribosylating enzyme that is responsible for the voluminous diarrhea characteristic of cholera disease. The genes encoding cholera toxin are located on the genome of the filamentous bacteriophage, CTXϕ, that integrates as a prophage into the V. cholerae chromosome. CTXϕ infection of V. cholerae requires the toxin-coregulated pilus and the periplasmic protein TolA. This infection process parallels that of Escherichia coli infection by the Ff family of filamentous coliphage. Here we demonstrate a direct interaction between the N-terminal domain of the CTXϕ minor coat protein pIII (pIII-N1) and the C-terminal domain of TolA (TolA-C) and present x-ray crystal structures of pIII-N1 alone and in complex with TolA-C. The structures of CTXϕ pIII-N1 and V. cholerae TolA-C are similar to coliphage pIII-N1 and E. coli TolA-C, respectively, yet these proteins bind via a distinct interface that in E. coli TolA corresponds to a colicin binding site. Our data suggest that the TolA binding site on pIII-N1 of CTXϕ is accessible in the native pIII protein. This contrasts with the Ff family phage, where the TolA binding site on pIII is blocked and requires a pilus-induced unfolding event to become exposed. We propose that CTXϕ pIII accesses the periplasmic TolA through retraction of toxin-coregulated pilus, which brings the phage through the outer membrane pilus secretin channel. These data help to explain the process by which CTXϕ converts a harmless marine microbe into a deadly human pathogen.

Francisella noatunensis subsp. noatunensis replicates within Atlantic cod (Gadus morhua L.) leucocytes and inhibits respiratory burst activity.

Francisella noatunensis subsp. noatunensis, causing granulomatosis in cod, has been shown to reside within cod immune cells, mainly within monocytes and macrophages. In the present study, we analysed the ability of the bacterium to replicate within adherent cells isolated from head kidney by in vitro infection of leucocytes. Two different technical approaches for flow cytometry analyses were performed for detection of intracellular bacteria. The presence of the wild type was assessed after identification by intracellular binding of specific antibodies to the pathogen. The other way was to use green fluorescent protein (GFP) transformed bacterium for infection studies allowing direct measurements of fluorescence from infected cells. By both methods we found an increase in fluorescence in infected cells, verifying bacterial replication, both after 4 and 28 h post infection in leucocytes isolated from head kidney (HKL). The GFP transformed bacterium was similar to the wild type in growth and infectivity pattern, showing that it can be a valuable tool for further studies of infection routes and pathology. Further, F. noatunensis subsp. noatunensis was found to inhibit respiratory burst activity, a potent pathogen killing mechanism, in cod leucocytes, but not in such cells from salmon. Our findings may indicate that inhibition of respiratory burst during Francisella infection is a key to its intracellular existence. This strategy seems to be conserved through evolution as it is also observed during infections in higher vertebrates caused by bacteria within the Francisella genus. The results presented here, showing the intracellular existence of Francisella, its replication within leucocytes and the inhibitory effect on respiratory burst, strongly support that these factors contribute to disease and pathology in infected cod. The intracellular replication shown in the present study might contribute to explain the problems of obtaining protective vaccines against Francisella and effective antibiotic treatment of infected fish.

Cohabitation of Piscirickettsia salmonis genogroups (LF-89 and EM-90): synergistic effect on growth dynamics.

Piscirickettsia salmonis, the biological agent of Salmonid Rickettsial Septicemia (SRS), is a facultative intracellular bacterium that can be divided into two genogroups (LF-89 and EM-90) with different virulence levels and patterns. Studies have found co-infection of these genogroups in salmonid farms in Chile, but it is essential to assess whether this interaction within the host is related to virulence and changes in pathogen dynamics. In this study, we studied four isolates from EM-90 and one LF-89 isolate chosen based on their genomic differences. The aim was to evaluate how co-cultivation affects bacterial growth performance and virulence factor expression in Atlantic salmon (Salmo salar) in vitro and in vivo. In vitro results using FN2 medium, showed a similar growth curve between co-cultures of LF-89 and EM-90 compared to EM-90 monocultures. This was explained by the higher ratio of EM-90 to LF-89 in all co-cultures. When evaluating the expression of virulence factors, it was discovered that the luxR gene was expressed only in EM-90-like isolates and that there were significant differences between mono- and co-cultures for flaA and cheA, suggesting a response to cohabitation. Moreover, during in vivo co-cultures, transcriptomic analysis revealed an upregulation of transposases, flagellum-related genes (fliI and flgK), transporters, and permeases that could unveil novel virulence effectors used in the early infection process of P. salmonis. Thus, our work has shown that cohabitation of P. salmonis genogroups can modulate their behavior and virulence effector expression. These data can contribute to new strategies and approaches to improve the current health treatments against this salmonid pathogen.

LitR of Vibrio salmonicida is a salinity-sensitive quorum-sensing regulator of phenotypes involved in host interactions and virulence.

Vibrio (Aliivibrio) salmonicida is the causal agent of cold-water vibriosis, a fatal bacterial septicemia primarily of farmed salmonid fish. The molecular mechanisms of invasion, colonization, and growth of V. salmonicida in the host are still largely unknown, and few virulence factors have been identified. Quorum sensing (QS) is a cell-to-cell communication system known to regulate virulence and other activities in several bacterial species. The genome of V. salmonicida LFI1238 encodes products presumably involved in several QS systems. In this study, the gene encoding LitR, a homolog of the master regulator of QS in V. fischeri, was deleted. Compared to the parental strain, the litR mutant showed increased motility, adhesion, cell-to-cell aggregation, and biofilm formation. Furthermore, the litR mutant produced less cryptic bioluminescence, whereas production of acylhomoserine lactones was unaffected. Our results also indicate a salinity-sensitive regulation of LitR. Finally, reduced mortality was observed in Atlantic salmon infected with the litR mutant, implying that the fish were more susceptible to infection with the wild type than with the mutant strain. We hypothesize that LitR inhibits biofilm formation and favors planktonic growth, with the latter being more adapted for pathogenesis in the fish host.

Vibrio salmonicida pathogenesis analyzed by experimental challenge of Atlantic salmon (Salmo salar).

Cold-water vibriosis (CV) is a bacterial septicemia of farmed salmonid fish and cod caused by the Gram-negative bacterium Vibrio (Aliivibrio) salmonicida. To study the pathogenesis of this marine pathogen, Atlantic salmon was experimentally infected by immersion challenge with wild type V. salmonicida and the bacterial distribution in different organs was investigated at different time points. V. salmonicida was identified in the blood as early as 2 h after challenge demonstrating a rapid establishment of bacteremia without an initial period of colonization of the host. Two days after immersion challenge, only a few V. salmonicida were identified in the intestines, but the amount increased with time. In prolonged CV cases, V. salmonicida was the dominating bacterium of the gut microbiota causing a release of the pathogen to the water. We hypothesize that V. salmonicida uses the blood volume for proliferation during the infection of the fish and the salmonid intestine as a reservoir that favors survival and transmission. In addition, a motility-deficient V. salmonicida strain led us to investigate the impact of motility in the CV pathogenesis by comparing the virulence properties of the mutant with the wild type LFI1238 strain in both i.p. and immersion challenge experiments. V. salmonicida was shown to be highly dependent on motility to gain access to the fish host. After invasion, motility was no longer required for virulence, but the absence of normal flagellation delayed the disease development.

Effects of different amoxicillin treatment durations on microbiome diversity and composition in the gut.

Antibiotics seize an effect on bacterial composition and diversity and have been demonstrated to induce disruptions on gut microbiomes. This may have implications for human health and wellbeing, and an increasing number of studies suggest a link between the gut microbiome and several diseases. Hence, reducing antibiotic treatments may be beneficial for human health status. Further, antimicrobial resistance (AMR) is an increasing global problem that can be counteracted by limiting the usage of antibiotics. Longer antibiotic treatments have been demonstrated to increase the development of AMR. Therefore, shortening of antibiotic treatment durations, provided it is safe for patients, may be one measure to reduce AMR. In this study, the objective was to investigate effects of standard and reduced antibiotic treatment lengths on gut microbiomes using a murine model. Changes in the murine gut microbiome was assessed after using three different treatment durations of amoxicillin (3, 7 or 14 days) as well as a control group not receiving amoxicillin. Fecal samples were collected before and during the whole experiment, until three weeks past end of treatment. These were further subject for 16S rRNA Illumina MiSeq sequencing. Our results demonstrated significant changes in bacterial diversity, richness and evenness during amoxicillin treatment, followed by a reversion in terms of alpha-diversity and abundance of major phyla, after end of treatment. However, a longer restitution time was indicated for mice receiving amoxicillin for 14 days, and phylum Patescibacteria did not fully recover. In addition, an effect on the composition of Firmicutes was indicated to last for at least three weeks in mice treated with amoxicillin for 14 days. Despite an apparently reversion to a close to original state in overall bacterial diversity and richness, the results suggested more durable changes in lower taxonomical levels. We detected several families, genera and ASVs with significantly altered abundance three weeks after exposure to amoxicillin, as well as bacterial taxa that appeared significantly affected by amoxicillin treatment length. This may strengthen the argument for shorter antibiotic treatment regimens to both limit the emergence of antibiotic resistance and risk of gut microbiome disturbance.

Global Regulatory Pathways Converge To Control Expression of Pseudomonas aeruginosa Type IV Pili.

The opportunistic pathogen Pseudomonas aeruginosa relies upon type IV pili (Tfp) for host colonization and virulence. Tfp are retractile surface appendages that promote adherence to host tissue and mediate twitching motility, a form of surface-associated translocation. Tfp are composed of a major structural pilin protein (PilA), several less abundant, fiber-associated pilin-like proteins (FimU, PilV, PilW, PilX, and PilE), and a pilus-associated tip adhesin and surface sensor (PilY1). Several proteins critical for Tfp biogenesis and surface sensing are encoded by the fimU-pilVWXY1Y2E operon. Tfp biogenesis is regulated by the global transcription factor Vfr and its allosteric effector, cyclic AMP (cAMP). Our investigation into the basis for reduced Tfp production in cAMP/vfr mutants revealed a defect in the expression of the fimU operon. We found that cAMP/Vfr activation of the fimU operon occurs via direct binding of Vfr to a specific fimU promoter sequence. We also refined the role of the AlgZ/AlgR two-component system in fimU regulation by demonstrating that phosphorylation of the response regulator AlgR is required for maximal binding to the fimU promoter region in vitro. Vfr also regulates expression of the algZR operon, revealing an indirect regulatory loop affecting fimU operon transcription. Overall, these results demonstrate that two linked but independent regulatory systems couple the expression of Tfp biogenesis and surface sensing genes and highlight the regulatory complexity governing expression of P. aeruginosa virulence factors. IMPORTANCE Pseudomonas aeruginosa is an opportunistic pathogen responsible for a wide range of infections. An extensive repertoire of virulence factors aid in P. aeruginosa pathogenesis. Type IV pili (Tfp) play a critical role in host colonization and infection by promoting adherence to host tissue, facilitating twitching motility and mediating surface-associated behaviors. The fimU operon encodes several pilus-associated proteins that are essential for proper Tfp function and surface sensing. In this study, we report that linked but independent regulatory systems dictate Tfp biogenesis. We also demonstrated the importance of different phosphorylation states of the AlgZ/AlgR two-component system and its role in Tfp biogenesis. Overall, this study furthers our understanding of the complex regulatory mechanisms that govern the production of a critical and multifaceted virulence factor.

O-linked glycosylation of the PilA pilin protein of Francisella tularensis: identification of the endogenous protein-targeting oligosaccharyltransferase and characterization of the native oligosaccharide.

Findings from a number of studies suggest that the PilA pilin proteins may play an important role in the pathogenesis of disease caused by species within the genus Francisella. As such, a thorough understanding of PilA structure and chemistry is warranted. Here, we definitively identified the PglA protein-targeting oligosaccharyltransferase by virtue of its necessity for PilA glycosylation in Francisella tularensis and its sufficiency for PilA glycosylation in Escherichia coli. In addition, we used mass spectrometry to examine PilA affinity purified from Francisella tularensis subsp. tularensis and F. tularensis subsp. holarctica and demonstrated that the protein undergoes multisite, O-linked glycosylation with a pentasaccharide of the structure HexNac-Hex-Hex-HexNac-HexNac. Further analyses revealed microheterogeneity related to forms of the pentasaccharide carrying unusual moieties linked to the distal sugar via a phosphate bridge. Type A and type B strains of Francisella subspecies thus express an O-linked protein glycosylation system utilizing core biosynthetic and assembly pathways conserved in other members of the proteobacteria. As PglA appears to be highly conserved in Francisella species, O-linked protein glycosylation may be a feature common to members of this genus.

Infection of human mucosal tissue by Pseudomonas aeruginosa requires sequential and mutually dependent virulence factors and a novel pilus-associated adhesin.

Tissue damage predisposes humans to life-threatening disseminating infection by the opportunistic pathogen Pseudomonas aeruginosa. Bacterial adherence to host tissue is a critical first step in this infection process. It is well established that P. aeruginosa attachment to host cells involves type IV pili (TFP), which are retractile surface fibres. The molecular details of attachment and the identity of the bacterial adhesin and host receptor remain controversial. Using a mucosal epithelium model system derived from primary human tissue, we show that the pilus-associated protein PilY1 is required for bacterial adherence. We establish that P. aeruginosa preferentially binds to exposed basolateral host cell surfaces, providing a mechanistic explanation for opportunistic infection of damaged tissue. Further, we demonstrate that invasion and fulminant infection of intact host tissue requires the coordinated and mutually dependent action of multiple bacterial factors, including pilus fibre retraction and the host cell intoxication system, termed type III secretion. Our findings offer new and important insights into the complex interactions between a pathogen and its human host and provide compelling evidence that PilY1 serves as the principal P. aeruginosa adhesin for human tissue and that it specifically recognizes a host receptor localized or enriched on basolateral epithelial cell surfaces.