Delivery of CdiA Nuclease Toxins into Target Cells during Contact-Dependent Growth Inhibition

Bacterial contact-dependent growth inhibition (CDI) is mediated by the CdiB/CdiA family of two-partner secretion proteins. CDI systems deploy a variety of distinct toxins, which are contained within the polymorphic C-terminal region (CdiA-CT) of CdiA proteins. Several CdiA-CTs are nucleases, suggesting that the toxins are transported into the target cell cytoplasm to interact with their substrates. To analyze CdiA transfer to target bacteria, we used the CDI system of uropathogenic Escherichia coli 536 (UPEC536) as a model. Antibodies recognizing the amino- and carboxyl-termini of CdiA^UPEC536 were used to visualize transfer of CdiA from CDI^UPEC536+ inhibitor cells to target cells using fluorescence microscopy. The results indicate that the entire CdiA^UPEC536 protein is deposited onto the surface of target bacteria. CdiA^UPEC536 transfer to bamA101 mutants is reduced, consistent with low expression of the CDI receptor BamA on these cells. Notably, our results indicate that the C-terminal CdiA-CT toxin region of CdiA^UPEC536 is translocated into target cells, but the N-terminal region remains at the cell surface based on protease sensitivity. These results suggest that the CdiA-CT toxin domain is cleaved from CdiA^UPEC536 prior to translocation. Delivery of a heterologous Dickeya dadantii CdiA-CT toxin, which has DNase activity, was also visualized. Following incubation with CDI⁺ inhibitor cells targets became anucleate, showing that the D.dadantii CdiA-CT was delivered intracellularly. Together, these results demonstrate that diverse CDI toxins are efficiently translocated across target cell envelopes.     

Genetic Analysis of the CDI Pathway from Burkholderia pseudomallei 1026b

Contact-dependent growth inhibition (CDI) is a mode of inter-bacterial competition mediated by the CdiB/CdiA family of two-partner secretion systems. CdiA binds to receptors on susceptible target bacteria, then delivers a toxin domain derived from its C-terminus. Studies with Escherichia coli suggest the existence of multiple CDI growth-inhibition pathways, whereby different systems exploit distinct target-cell proteins to deliver and activate toxins. Here, we explore the CDI pathway in Burkholderia using the CDI_II ^Bp1026b system encoded on chromosome II of Burkholderia pseudomallei 1026b as a model. We took a genetic approach and selected Burkholderia thailandensis E264 mutants that are resistant to growth inhibition by CDI_II ^Bp1026b. We identified mutations in three genes, BTH_I0359, BTH_II0599, and BTH_I0986, each of which confers resistance to CDI_II ^Bp1026b. BTH_I0359 encodes a small peptide of unknown function, whereas BTH_II0599 encodes a predicted inner membrane transport protein of the major facilitator superfamily. The inner membrane localization of BTH_II0599 suggests that it may facilitate translocation of CdiA-CT_II ^Bp1026b toxin from the periplasm into the cytoplasm of target cells. BTH_I0986 encodes a putative transglycosylase involved in lipopolysaccharide (LPS) synthesis. ∆BTH_I0986 mutants have altered LPS structure and do not interact with CDI⁺ inhibitor cells to the same extent as BTH_I0986⁺ cells, suggesting that LPS could function as a receptor for CdiA_II ^Bp1026b. Although ∆BTH_I0359, ∆BTH_II0599, and ∆BTH_I0986 mutations confer resistance to CDI_II ^Bp1026b, they provide no protection against the CDI^E264 system deployed by B. thailandensis E264. Together, these findings demonstrate that CDI growth-inhibition pathways are distinct and can differ significantly even between closely related species.     

Kind Discrimination and Competitive Exclusion Mediated by Contact-Dependent Growth Inhibition Systems Shape Biofilm Community Structure

Contact-Dependent Growth Inhibition (CDI) is a phenomenon in which bacteria use the toxic C-terminus of a large exoprotein (called BcpA in Burkholderia species) to inhibit the growth of neighboring bacteria upon cell-cell contact. CDI systems are present in a wide range of Gram-negative proteobacteria and a hallmark feature is polymorphism amongst the exoprotein C-termini (BcpA-CT in Burkholderia) and amongst the small immunity proteins (BcpI) that protect against CDI in an allele-specific manner. In addition to CDI, the BcpAIOB proteins of Burkholderia thailandensis mediate biofilm formation, and they do so independent of BcpA-mediated interbacterial competition, suggesting a cooperative role for CDI system proteins in this process. CDI has previously only been demonstrated between CDI⁺ and CDI⁻ bacteria, leaving the roles of CDI system-mediated interbacterial competition and of CDI system diversity in nature unknown. We constructed B. thailandensis strains that differed only in the BcpA-CT and BcpI proteins they produced. When co-cultured on agar, these strains each participated in CDI and the outcome of the competition depended on both CDI system efficiency and relative bacterial numbers initially. Strains also participated in CDI during biofilm development, resulting in pillar structures that were composed of only a single BcpA-CT/BcpI type. Moreover, a strain producing BcpA-CT/BcpI proteins of one type was prevented from joining a pre-established biofilm community composed of bacteria producing BcpA-CT/BcpI proteins of a different type, unless it also produced the BcpI protein of the established strain. Bacteria can therefore use CDI systems for kind recognition and competitive exclusion of ‘non-self’ bacteria from a pre-established biofilm. Our data indicate that CDI systems function in both cooperative and competitive behaviors to build microbial communities that are composed of only bacteria that are related via their CDI system alleles.     

The Cytoplasm-Entry Domain of Antibacterial CdiA Is a Dynamic α-Helical Bundle with Disulfide-Dependent Structural Features

Many Gram‐negative bacterial species use contact-dependent growth inhibition (CDI) systems to compete with neighboring cells. CDI⁺ strains express cell-surface CdiA effector proteins, which carry a toxic C-terminal region (CdiA-CT) that is cleaved from the effector upon transfer into the periplasm of target bacteria. The released CdiA-CT consists of two domains. The C-terminal domain is typically a nuclease that inhibits cell growth, and the N-terminal “cytoplasm-entry” domain mediates toxin translocation into the target-cell cytosol. Here, we use NMR and circular dichroism spectroscopic approaches to probe the structure, stability, and dynamics of the cytoplasm-entry domain from Escherichia coli STEC_MHI813. Chemical shift analysis reveals that the CdiA-CT^MHI813 entry domain is composed of a C-terminal helical bundle and a dynamic N-terminal region containing two disulfide linkages. Disruption of the disulfides by mutagenesis or chemical reduction destabilizes secondary structure over the N-terminus, but has no effect on the C-terminal helices. Although critical for N-terminal structure, the disulfides have only modest effects on global thermodynamic stability, and the entry domain exhibits characteristics of a molten globule. We find that the disulfides form in vivo as the entry domain dwells in the periplasm of inhibitor cells prior to target-cell recognition. CdiA-CT^MHI813 variants lacking either disulfide still kill target bacteria, but disruption of both bonds abrogates growth inhibition activity. We propose that the entry domain's dynamic structural features are critical for function. In its molten globule-like state, the domain resists degradation after delivery, yet remains pliable enough to unfold for membrane translocation.     

Identification of functional toxin/immunity genes linked to contact-dependent growth inhibition (CDI) and rearrangement hotspot (Rhs) systems

Bacterial contact-dependent growth inhibition (CDI) is mediated by the CdiA/CdiB family of two-partner secretion proteins. Each CdiA protein exhibits a distinct growth inhibition activity, which resides in the polymorphic C-terminal region (CdiA-CT). CDI(+) cells also express unique CdiI immunity proteins that specifically block the activity of cognate CdiA-CT, thereby protecting the cell from autoinhibition. Here we show that many CDI systems contain multiple cdiA gene fragments that encode CdiA-CT sequences. These "orphan" cdiA-CT genes are almost always associated with downstream cdiI genes to form cdiA-CT/cdiI modules. Comparative genome analyses suggest that cdiA-CT/cdiI modules are mobile and exchanged between the CDI systems of different bacteria. In many instances, orphan cdiA-CT/cdiI modules are fused to full-length cdiA genes in other bacterial species. Examination of cdiA-CT/cdiI modules from Escherichia coli EC93, E. coli EC869, and Dickeya dadantii 3937 confirmed that these genes encode functional toxin/immunity pairs. Moreover, the orphan module from EC93 was functional in cell-mediated CDI when fused to the N-terminal portion of the EC93 CdiA protein. Bioinformatic analyses revealed that the genetic organization of CDI systems shares features with rhs (rearrangement hotspot) loci. Rhs proteins also contain polymorphic C-terminal regions (Rhs-CTs), some of which share significant sequence identity with CdiA-CTs. All rhs genes are followed by small ORFs representing possible rhsI immunity genes, and several Rhs systems encode orphan rhs-CT/rhsI modules. Analysis of rhs-CT/rhsI modules from D. dadantii 3937 demonstrated that Rhs-CTs have growth inhibitory activity, which is specifically blocked by cognate RhsI immunity proteins. Together, these results suggest that Rhs plays a role in intercellular competition and that orphan gene modules expand the diversity of toxic activities deployed by both CDI and Rhs systems.     

Contact-Dependent Growth Inhibition Causes Reversible Metabolic Downregulation in Escherichia coli

Contact-dependent growth inhibition (CDI) is a mechanism identified in Escherichia coli by which bacteria expressing two-partner secretion proteins encoded by cdiA and cdiB bind to BamA in the outer membranes of target cells and inhibit their growth. A third gene in the cluster, cdiI, encodes a small protein that is necessary and sufficient to confer immunity to CDI, thereby preventing cells expressing the cdiBA genes from inhibiting their own growth. In this study, the cdiI gene was placed under araBAD promoter control to modulate levels of the immunity protein and thereby induce CDI by removal of arabinose. This CDI autoinhibition system was used for metabolic analyses of a single population of E. coli cells undergoing CDI. Contact-inhibited cells showed altered cell morphology, including the presence of filaments. Notably, CDI was reversible, as evidenced by resumption of cell growth and normal cellular morphology following induction of the CdiI immunity protein. Recovery of cells from CDI also required an energy source. Cells undergoing CDI showed a significant, reversible downregulation of metabolic parameters, including aerobic respiration, proton motive force (Δp), and steady-state ATP levels. It is unclear whether the decrease in respiration and/or Δp is directly involved in growth inhibition, but a role for ATP in the CDI mechanism was ruled out using an atp mutant. Consistent with the observed decrease in Δp, the phage shock response was induced in cells undergoing CDI but not in recovering cells, based on analysis of levels of pspA mRNA.Intercellular communication mechanisms enable bacteria to coordinate biological phenomena such as DNA uptake, differentiation for fruiting body development, light production, and swarming (7, 8). These cell-to-cell interactions enable individual bacteria to form a multicellular community, such as in a biofilm on a solid surface, under specific environmental conditions (20, 52). Similarly to multicellular organisms, bacteria have signal transduction mechanisms to facilitate cellular cross talk, including two-component regulatory systems and other cell surface ligand-receptor interactions that control cellular processes.We previously described a cross talk phenomenon designated as contact-dependent growth inhibition (CDI) in which one bacterial isolate (CDI⁺) blocks the growth of another bacterium when mixed together (4). CDI requires two contiguous genes, cdiB and cdiA, which encode proteins that are in the two-partner secretion (TPS) family. Overlapping the stop codon of cdiA is a downstream open reading frame designated cdiI, which encodes a 79-amino-acid protein that provides immunity to growth inhibition from cells expressing cdiBA (4). Evidence strongly indicates that cell-to-cell contact is required for growth inhibition. First, separation of CDI⁺ inhibitor cells and target cells by a 0.4-μm nitrocellulose membrane blocked CDI, distinguishing CDI from the class of soluble bacterial growth inhibitors known as bacteriocins. Second, to address the possibility that a very short-lived bacteriocin-like molecule might be released from CDI⁺ inhibitor cells, we separated inhibitor-target cell aggregates by fluorescence-activated cell sorting. Target cells within aggregates with CDI⁺ inhibitor cells lost viability, as measured by growth on LB medium, more rapidly than did unbound target cells, supporting the conclusion that cell-to-cell contact is required for CDI (4).In our previous work, analysis of CDI was carried out with a bipartite system using Escherichia coli inhibitor cells containing cdiB, -A, and -I on a multicopy plasmid that constitutively expressed CDI activity, cocultured with E. coli K-12 target cells. Mixing inhibitor cells with E. coli K-12 target cells resulted in a 5- to 6-log decrease in target cell number after only 1 to 2 h (4). Because this bipartite CDI assay contains both inhibitor and target cells, monitoring of target cell growth in real time is not possible. Thus, we have not been able to determine if CDI is a reversible process or a nonreversible toxin-like system. This is important in assessing the role of CDI in the biology of E. coli as well as its potential role in many gram-negative bacteria, including uropathogenic E. coli, Burkholderia pseudomallei, and Yersinia pestis that contain genes with significant sequence identity to cdiB and cdiA (4).Recently, in collaboration with J. Malinverni and T. Silhavy (Princeton University), we showed that the target cell receptor for CDI is BamA, an essential outer membrane protein (OMP). Homologues of bamA are conserved in genomes from bacteria to mitochondria (3). BamA is a key component of the β-barrel assembly machine required for biogenesis of many other OMPs (34, 43, 53). Our results indicated that the BamA receptor facilitates CdiB/CdiA-dependent cell-to-cell binding and growth inhibition since antibodies to BamA blocked formation of inhibitor-target cell aggregates and CDI (3). The ligand for BamA is not known, but it seems probable that it is CdiA, which is at the surface of inhibitor cells (4), and may form a short 40- to 50-nm fiber, such as filamentous hemagglutinin in Bordetella pertussis, based on sequence similarities (42). AcrB is also required for sensitivity of target cells to CDI, which acts downstream of the BamA receptor (3). AcrB is a protein that exports small molecules, including drugs/antibiotics, through the inner membrane, with further transport through the outer membrane in conjunction with AcrA and TolC (47, 55, 56). This export machine requires proton motive force (Δp) as an energy source (46). Markedly, only BamA and AcrB, independent from their respective export machines, are required for CDI (3). Based on these results, we developed a model in which CdiA of inhibitor cells bind to BamA on target cells, transmitting a signal into target cells such as a CdiA peptide (Fig. ​(Fig.1A).1A). This signal could enter cells through an AcrB portal to interact with an unidentified cytosolic target. It is also possible that AcrB could play an indirect role in CDI-mediated growth inhibition.Open in a separate windowFIG. 1.Construction of a contact-dependent autoinhibition system in Escherichia coli. (A) CDI model. CdiB and CdiA (CdiBA) expressed by one cell (inhibitor cell) binds to BamA in the outer membrane (OM) of an adjacent cell (target cell) (3). A signal, such as a CdiA peptide, is transferred through BamA to AcrB located in the inner membrane (IM), causing inhibition of cell growth by an unknown mechanism. CDI is modulated at the cell surface by certain pili, including pyelonephritis-associated pili (4), and by colanic acid capsule (3) and inside cells by CdiI immunity protein (4). (B) Map of the CDI autoinhibition plasmid (pDAL728).Here, we describe the development of a CDI autoinhibition system in which growth inhibition is regulated by controlled expression of the CdiI immunity protein, enabling examination of a single population of cells undergoing CDI. Using this system, we show that cells undergoing CDI have significantly reduced cellular respiration, Δp, and steady-state ATP levels. Notably, this metabolic downregulation, as well as cellular growth inhibition, is reversible. To the best of our knowledge, this is the first report of a natural, reversible system controlling bacterial metabolism and growth.     

Biofilm regulation in Clostridioides difficile: Novel systems linked to hypervirulence

Clostridiodes difficile (C. difficile) was ranked an “urgent threat” by the Centers for Disease Control and Prevention (CDC) in 2019. C. difficile infection (CDI) is the most common healthcare-associated infection (HAI) in the United States of America as well as the leading cause of antibiotic-associated gastrointestinal disease. C. difficile is a gram-positive, rod-shaped, spore-forming, anaerobic bacterium that causes infection of the epithelial lining of the gut. CDI occurs most commonly after disruption of the human gut microflora following the prolonged use of broad-spectrum antibiotics. However, the recurrent nature of this disease has led to the hypothesis that biofilm formation may play a role in its pathogenesis. Biofilms are sessile communities of bacteria protected from extracellular stresses by a matrix of self-produced proteins, polysaccharides, and extracellular DNA. Biofilm regulation in C. difficile is still incompletely understood, and its role in disease recurrence has yet to be fully elucidated. However, many factors have been found to influence biofilm formation in C. difficile, including motility, adhesion, and hydrophobicity of the bacterial cells. Small changes in one of these systems can greatly influence biofilm formation. Therefore, the biofilm regulatory system would need to coordinate all these systems to create optimal biofilm-forming physiology under appropriate environmental conditions. The coordination of these systems is complex and multifactorial, and any analysis must take into consideration the influences of the stress response, quorum sensing (QS), and gene regulation by second messenger molecule cyclic diguanosine monophosphate (c-di-GMP). However, the differences in biofilm-forming ability between C. difficile strains such as 630 and the “hypervirulent” strain, {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291, make it difficult to assign a “one size fits all” mechanism to biofilm regulation in C. difficile. This review seeks to consolidate published data regarding the regulation of C. difficile biofilms in order to identify gaps in knowledge and propose directions for future study.     

Flagellin Treatment Prevents Increased Susceptibility to Systemic Bacterial Infection after Injury by Inhibiting Anti-Inflammatory IL-10+ IL-12- Neutrophil Polarization

Severe trauma renders patients susceptible to infection. In sepsis, defective bacterial clearance has been linked to specific deviations in the innate immune response. We hypothesized that innate immune modulations observed during sepsis also contribute to increased bacterial susceptibility after severe trauma. A well-established murine model of burn injury, used to replicate infection following trauma, showed that wound inoculation with P. aeruginosa quickly spreads systemically. The systemic IL-10/IL-12 axis was skewed after burn injury with infection as indicated by a significant elevation in serum IL-10 and polarization of neutrophils into an anti-inflammatory (“N2”; IL-10⁺ IL-12⁻) phenotype. Infection with an attenuated P. aeruginosa strain (ΔCyaB) was cleared better than the wildtype strain and was associated with an increased pro-inflammatory neutrophil (“N1”; IL-10⁻IL-12⁺) response in burn mice. This suggests that neutrophil polarization influences bacterial clearance after burn injury. Administration of a TLR5 agonist, flagellin, after burn injury restored the neutrophil response towards a N1 phenotype resulting in an increased clearance of wildtype P. aeruginosa after wound inoculation. This study details specific alterations in innate cell populations after burn injury that contribute to increased susceptibility to bacterial infection. In addition, for the first time, it identifies neutrophil polarization as a therapeutic target for the reversal of bacterial susceptibility after injury.     

Infection Density Dynamics of the Citrus Greening Bacterium “Candidatus Liberibacter asiaticus” in Field Populations of the Psyllid Diaphorina citri and Its Relevance to the Efficiency of Pathogen Transmission to Citrus Plants

Huanglongbing, or citrus greening, is a devastating disease of citrus plants recently spreading worldwide, which is caused by an uncultivable bacterial pathogen, “Candidatus Liberibacter asiaticus,” and vectored by a phloem-sucking insect, Diaphorina citri. We investigated the infection density dynamics of “Ca. Liberibacter asiaticus” in field populations of D. citri with experiments using field-collected insects to address how “Ca. Liberibacter asiaticus” infection density in the vector insect is relevant to pathogen transmission to citrus plants. Of 500 insects continuously collected from “Ca. Liberibacter asiaticus”-infected citrus trees with pathological symptoms in the spring and autumn of 2009, 497 (99.4%) were “Ca. Liberibacter asiaticus” positive. The infections were systemic across head-thorax and abdomen, ranging from 10³ to 10⁷ bacteria per insect. In spring, the infection densities were low in March, at ∼10³ bacteria per insect, increasing up to 10⁶ to 10⁷ bacteria per insect in April and May, and decreasing to 10⁵ to 10⁶ bacteria per insect in late May, whereas the infection densities were constantly ∼10⁶ to 10⁷ bacteria per insect in autumn. Statistical analysis suggested that several factors, such as insect sex, host trees, and collection dates, may be correlated with “Ca. Liberibacter asiaticus” infection densities in field D. citri populations. Inoculation experiments with citrus seedlings using field-collected “Ca. Liberibacter asiaticus”-infected insects suggested that (i) “Ca. Liberibacter asiaticus”-transmitting insects tend to exhibit higher infection densities than do nontransmitting insects, (ii) a threshold level (∼10⁶ bacteria per insect) of “Ca. Liberibacter asiaticus” density in D. citri is required for successful transmission to citrus plants, and (iii) D. citri attaining the threshold infection level transmits “Ca. Liberibacter asiaticus” to citrus plants in a stochastic manner. These findings provide valuable insights into understanding, predicting, and controlling this notorious citrus pathogen.     

Antimicrobial Activity of Human Prion Protein Is Mediated by Its N-Terminal Region

Background

Cellular prion-related protein (PrP^c) is a cell-surface protein that is ubiquitously expressed in the human body. The multifunctionality of PrP^c, and presence of an exposed cationic and heparin-binding N-terminus, a feature characterizing many antimicrobial peptides, made us hypotesize that PrP^c could exert antimicrobial activity.

Methodology and Principal Findings

Intact recombinant PrP exerted antibacterial and antifungal effects at normal and low pH. Studies employing recombinant PrP and N- and C-terminally truncated variants, as well as overlapping peptide 20mers, demonstrated that the antimicrobial activity is mediated by the unstructured N-terminal part of the protein. Synthetic peptides of the N-terminus of PrP killed the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, and the Gram-positive Bacillus subtilis and Staphylococcus aureus, as well as the fungus Candida parapsilosis. Fluorescence studies of peptide-treated bacteria, paired with analysis of peptide effects on liposomes, showed that the peptides exerted membrane-breaking effects similar to those seen after treatment with the “classical” human antimicrobial peptide LL-37. In contrast to LL-37, however, no marked helix induction was detected for the PrP-derived peptides in presence of negatively charged (bacteria-mimicking) liposomes. PrP furthermore showed an inducible expression during wounding of human skin ex vivo and in vivo, as well as stimulation of keratinocytes with TGF-α in vitro.

Conclusions

The demonstration of an antimicrobial activity of PrP, localisation of its activity to the N-terminal and heparin-binding region, combined with results showing an increased expression of PrP during wounding, indicate that PrPs could have a previously undisclosed role in host defense.     

Structural Basis for Type VI Secretion Effector Recognition by a Cognate Immunity Protein

The type VI secretion system (T6SS) has emerged as an important mediator of interbacterial interactions. A T6SS from Pseudomonas aeruginosa targets at least three effector proteins, type VI secretion exported 1–3 (Tse1–3), to recipient Gram-negative cells. The Tse2 protein is a cytoplasmic effector that acts as a potent inhibitor of target cell proliferation, thus providing a pronounced fitness advantage for P. aeruginosa donor cells. P. aeruginosa utilizes a dedicated immunity protein, type VI secretion immunity 2 (Tsi2), to protect against endogenous and intercellularly-transferred Tse2. Here we show that Tse2 delivered by the T6SS efficiently induces quiescence, not death, within recipient cells. We demonstrate that despite direct interaction of Tsi2 and Tse2 in the cytoplasm, Tsi2 is dispensable for targeting the toxin to the secretory apparatus. To gain insights into the molecular basis of Tse2 immunity, we solved the 1.00 Å X-ray crystal structure of Tsi2. The structure shows that Tsi2 assembles as a dimer that does not resemble previously characterized immunity or antitoxin proteins. A genetic screen for Tsi2 mutants deficient in Tse2 interaction revealed an acidic patch distal to the Tsi2 homodimer interface that mediates toxin interaction and immunity. Consistent with this finding, we observed that destabilization of the Tsi2 dimer does not impact Tse2 interaction. The molecular insights into Tsi2 structure and function garnered from this study shed light on the mechanisms of T6 effector secretion, and indicate that the Tse2–Tsi2 effector–immunity pair has features distinguishing it from previously characterized toxin–immunity and toxin–antitoxin systems.     

Phylogenetic Characterization of Two Novel Commensal Bacteria Involved with Innate Immune Homeostasis in Drosophila melanogaster

During a previous study on the molecular interaction between commensal bacteria and host gut immunity, two novel bacterial strains, A911^T and G707^T, were isolated from the gut of Drosophila melanogaster. In this study, these strains were characterized in a polyphasic taxonomic study using phenotypic, genetic, and chemotaxonomic analyses. We show that the strains represent novel species in the family Acetobacteraceae. Strain G707^T, a highly pathogenic organism, represents a new species in the genus Gluconobacter, “Gluconobacter morbifer” sp. nov. (type strain G707 = KCTC 22116^T = JCM 15512^T). Strain A911^T, dominantly present in the normal Drosphila gut community, represents a novel genus and species, designated “Commensalibacter intestini” gen. nov., sp. nov. (type strain A911 = KCTC 22117^T = JCM 15511^T).     

The Periplasmic Protein TolB as a Potential Drug Target in Pseudomonas aeruginosa

The Gram-negative bacterium Pseudomonas aeruginosa is one of the most dreaded pathogens in the hospital setting, and represents a prototype of multi-drug resistant “superbug” for which effective therapeutic options are very limited. The identification and characterization of new cellular functions that are essential for P. aeruginosa viability and/or virulence could drive the development of anti-Pseudomonas compounds with novel mechanisms of action. In this study we investigated whether TolB, the periplasmic component of the Tol-Pal trans-envelope protein complex of Gram-negative bacteria, represents a potential drug target in P. aeruginosa. By combining conditional mutagenesis with the analysis of specific pathogenicity-related phenotypes, we demonstrated that TolB is essential for P. aeruginosa growth, both in laboratory and clinical strains, and that TolB-depleted P. aeruginosa cells are strongly defective in cell-envelope integrity, resistance to human serum and several antibiotics, as well as in the ability to cause infection and persist in an insect model of P. aeruginosa infection. The essentiality of TolB for P. aeruginosa growth, resistance and pathogenicity highlights the potential of TolB as a novel molecular target for anti-P. aeruginosa drug discovery.     

Virulence Factors of Pseudomonas aeruginosa Induce Both the Unfolded Protein and Integrated Stress Responses in Airway Epithelial Cells

Pseudomonas aeruginosa infection can be disastrous in chronic lung diseases such as cystic fibrosis and chronic obstructive pulmonary disease. Its toxic effects are largely mediated by secreted virulence factors including pyocyanin, elastase and alkaline protease (AprA). Efficient functioning of the endoplasmic reticulum (ER) is crucial for cell survival and appropriate immune responses, while an excess of unfolded proteins within the ER leads to “ER stress” and activation of the “unfolded protein response” (UPR). Bacterial infection and Toll-like receptor activation trigger the UPR most likely due to the increased demand for protein folding of inflammatory mediators. In this study, we show that cell-free conditioned medium of the PAO1 strain of P. aeruginosa, containing secreted virulence factors, induces ER stress in primary bronchial epithelial cells as evidenced by splicing of XBP1 mRNA and induction of CHOP, GRP78 and GADD34 expression. Most aspects of the ER stress response were dependent on TAK1 and p38 MAPK, except for the induction of GADD34 mRNA. Using various mutant strains and purified virulence factors, we identified pyocyanin and AprA as inducers of ER stress. However, the induction of GADD34 was mediated by an ER stress-independent integrated stress response (ISR) which was at least partly dependent on the iron-sensing eIF2α kinase HRI. Our data strongly suggest that this increased GADD34 expression served to protect against Pseudomonas-induced, iron-sensitive cell cytotoxicity. In summary, virulence factors from P. aeruginosa induce ER stress in airway epithelial cells and also trigger the ISR to improve cell survival of the host.     

Characterization of a New Bacteriocin Operon in Sakacin P-Producing Lactobacillus sakei, Showing Strong Translational Coupling between the Bacteriocin and Immunity Genes

Previous studies of genes involved in the production of sakacin P by Lactobacillus sakei Lb674 revealed the presence of an inducible promoter downstream of the known spp gene clusters. We show here that this promoter drives the expression of an operon consisting of a bacteriocin gene (sppQ), a cognate immunity gene (spiQ), another gene with an unknown function (orf4), and a pseudoimmunity gene containing a frameshift mutation (orf5). The leader peptide of the new one-peptide bacteriocin sakacin Q contains consensus elements that are typical for so-called “double-glycine” leader peptides. The mature bacteriocin shows weak similarity to the BrcA peptide of the two-peptide bacteriocin brochocin C. Sakacin Q has an antimicrobial spectrum that differs from that of sakacin P, thus expanding the antimicrobial properties of the producer strain. The genes encoding sakacin Q and its cognate immunity protein showed strong translational coupling, which was investigated in detail by analyzing the properties of a series of β-glucuronidase fusions. Our results provide experimental evidence that production of the bacteriocin and production of the cognate immunity protein are tightly coregulated at the translational level.     

XoxF-Type Methanol Dehydrogenase from the Anaerobic Methanotroph “Candidatus Methylomirabilis oxyfera”

“Candidatus Methylomirabilis oxyfera” is a newly discovered anaerobic methanotroph that, surprisingly, oxidizes methane through an aerobic methane oxidation pathway. The second step in this aerobic pathway is the oxidation of methanol. In Gram-negative bacteria, the reaction is catalyzed by pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenase (MDH). The genome of “Ca. Methylomirabilis oxyfera” putatively encodes three different MDHs that are localized in one large gene cluster: one so-called MxaFI-type MDH and two XoxF-type MDHs (XoxF1 and XoxF2). MxaFI MDHs represent the canonical enzymes, which are composed of two PQQ-containing large (α) subunits (MxaF) and two small (β) subunits (MxaI). XoxF MDHs are novel, ecologically widespread, but poorly investigated types of MDHs that can be phylogenetically divided into at least five different clades. The XoxF MDHs described thus far are homodimeric proteins containing a large subunit only. Here, we purified a heterotetrameric MDH from “Ca. Methylomirabilis oxyfera” that consisted of two XoxF and two MxaI subunits. The enzyme was localized in the periplasm of “Ca. Methylomirabilis oxyfera” cells and catalyzed methanol oxidation with appreciable specific activity and affinity (V_max of 10 μmol min⁻¹ mg⁻¹ protein, K_m of 17 μM). PQQ was present as the prosthetic group, which has to be taken up from the environment since the known gene inventory required for the synthesis of this cofactor is lacking. The MDH from “Ca. Methylomirabilis oxyfera” is the first representative of type 1 XoxF proteins to be described.     

Caspase-1-driven neutrophil pyroptosis and its role in host susceptibility to Pseudomonas aeruginosa

Multiple regulated neutrophil cell death programs contribute to host defense against infections. However, despite expressing all necessary inflammasome components, neutrophils are thought to be generally defective in Caspase-1-dependent pyroptosis. By screening different bacterial species, we found that several Pseudomonas aeruginosa (P. aeruginosa) strains trigger Caspase-1-dependent pyroptosis in human and murine neutrophils. Notably, deletion of Exotoxins U or S in P. aeruginosa enhanced neutrophil death to Caspase-1-dependent pyroptosis, suggesting that these exotoxins interfere with this pathway. Mechanistically, P. aeruginosa Flagellin activates the NLRC4 inflammasome, which supports Caspase-1-driven interleukin (IL)-1β secretion and Gasdermin D (GSDMD)-dependent neutrophil pyroptosis. Furthermore, P. aeruginosa-induced GSDMD activation triggers Calcium-dependent and Peptidyl Arginine Deaminase-4-driven histone citrullination and translocation of neutrophil DNA into the cell cytosol without inducing extracellular Neutrophil Extracellular Traps. Finally, we show that neutrophil Caspase-1 contributes to IL-1β production and susceptibility to pyroptosis-inducing P. aeruginosa strains in vivo. Overall, we demonstrate that neutrophils are not universally resistant for Caspase-1-dependent pyroptosis.     

Procalcitonin Levels Associate with Severity of Clostridium difficile Infection

Objective

Clostridium difficile infection (CDI) is a major cause of morbidity and biomarkers that predict severity of illness are needed. Procalcitonin (PCT), a serum biomarker with specificity for bacterial infections, has been little studied in CDI. We hypothesized that PCT associated with CDI severity.

Design

Serum PCT levels were measured for 69 cases of CDI. Chart review was performed to evaluate the presence of severity markers and concurrent acute bacterial infection (CABI). We defined the binary variables clinical score as having fever (T >38°C), acute organ dysfunction (AOD), and/or WBC >15,000 cells/mm³ and expanded score, which included the clinical score plus the following: ICU admission, no response to therapy, colectomy, and/or death.

Results

In univariate analysis log₁₀ PCT associated with clinical score (OR 3.13, 95% CI 1.69–5.81, P<.001) and expanded score (OR 3.33, 95% CI 1.77–6.23, P<.001). In a multivariable model including the covariates log₁₀ PCT, enzyme immunoassay for toxin A/B, ribotype 027, age, weighted Charlson-Deyo comorbidity index, CABI, and extended care facility residence, log₁₀ PCT associated with clinical score (OR 3.09, 95% CI 1.5–6.35, P = .002) and expanded score (OR 3.06, 95% CI 1.49–6.26, P = .002). PCT >0.2 ng/mL was 81% sensitive/73% specific for a positive clinical score and had a negative predictive value of 90%.

Conclusion

An elevated PCT level associated with the presence of CDI severity markers and CDI was unlikely to be severe with a serum PCT level below 0.2 ng/mL. The extent to which PCT changes during CDI therapy or predicts recurrent CDI remains to be quantified.