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Salmonella enterica serovar Typhimurium is a facultative intracellular pathogen that invades the intestinal epithelium. Following invasion of epithelial cells, Salmonella survives and replicates within two distinct intracellular niches. While all of the bacteria are initially taken up into a membrane bound vacuole, the Salmonella-containing vacuole or SCV, a significant proportion of them promptly escape into the cytosol. Cytosolic Salmonella replicates more rapidly compared to the vacuolar population, although the reasons for this are not well understood. SipA, a multi-function effector protein, has been shown to affect intracellular replication and is secreted by cytosolic Salmonella via the invasion-associated Type III Secretion System 1 (T3SS1). Here, we have used a multipronged microscopy approach to show that SipA does not affect bacterial replication rates per se, but rather mediates intra-cytosolic survival and/or initiation of replication following bacterial egress from the SCV. Altogether, our findings reveal an important role for SipA in the early survival of cytosolic Salmonella., (© 2019 John Wiley & Sons Ltd.)

Xenophagy, the process of eliminating intracellular pathogens through the autophagy machinery, is an important defense mechanism against infectious disease, yet the underlying molecular mechanisms remain incompletely understood. Recent work (Xu et al., Cell, 2019) used the discovery of an inhibitor of xenophagy, SopF, to identify a SopF-sensitive mechanism that allows mammalian cells to detect invading bacteria., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Mycobacterium tuberculosis (Mtb) protein tyrosine phosphatase (PtpA) has so far been known to control intracellular survival of mycobacteria; whereas the ATP synthase which is essential for mycobacterial growth has recently been contemplated in developing a breakthrough anti-TB drug, diarylquinoline. Since both of these enzymes have been established as validated drug targets; we report a robust and functional relationship between these two enzymes through a series of experiments using Mtb H37Ra. In the present study we report that the mycobacterial ATP synthase alpha subunit is regulated by PtpA. We generated gene knock-out for the enzyme PtpA and subjected to determine the mycobacterial replication and the proteome profile of wild type, mutant (ΔptpA) and complemented (ΔptpA:ptpA) strains of Mtb H37Ra. A substantial amount of decrease in the protein level of ATP synthase alpha subunit (AtpA) in case of mutant H37Ra was observed, while the levels of the enzyme were either increased or remained unchanged, in wild type and in the complemented strains., (Copyright © 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Biological water oxidation, performed by a single enzyme, photosystem II, is a central research topic not only in understanding the photosynthetic apparatus but also for the development of water splitting catalysts for technological applications. Great progress has been made in this endeavor following the report of a high-resolution X-ray crystallographic structure in 2011 resolving the cofactor site (Umena et al. in Nature 473:55-60, 2011), a tetra-manganese calcium complex. The electronic properties of the protein-bound water oxidizing Mn 4 O x Ca complex are crucial to understand its catalytic activity. These properties include: its redox state(s) which are tuned by the protein matrix, the distribution of the manganese valence and spin states and the complex interactions that exist between the four manganese ions. In this short review we describe how magnetic resonance techniques, particularly EPR, complemented by quantum chemical calculations, have played an important role in understanding the electronic structure of the cofactor. Together with isotope labeling, these techniques have also been instrumental in deciphering the binding of the two substrate water molecules to the cluster. These results are briefly described in the context of the history of biological water oxidation with special emphasis on recent work using time resolved X-ray diffraction with free electron lasers. It is shown that these data are instrumental for developing a model of the biological water oxidation cycle.

Bacterial cell division is initiated by tubulin homologue FtsZ that assembles into a ring structure at mid-cell to facilitate cytokinesis. EzrA has been identified to be implicated in FtsZ-ring dynamics and cell wall biosynthesis during cell division of Bacillus subtilis and Staphylococcus aureus, the model rod and cocci. However, its role in pathogenic streptococci remains largely unknown. Here, the role of EzrA was investigated in Streptococcus mutans, the primary etiological agent of human dental caries, by constructing an ezrA in-frame deletion mutant. Our data showed that the ezrA mutant was slow-growing with a shortened length and extended width round cell shape compared to the wild type, indicating a delay in cell division with abnormalities of peptidoglycan biosynthesis. Additionally, FtsZ irregularly localized in dividing ezrA mutant cells forming angled division planes, potentially contributing to an aberrant cell shape. Furthermore, investigation using single-species cariogenic biofilm model revealed that deletion of ezrA resulted in defective biofilm formation with less extracellular polysaccharides and altered three-dimensional biofilm architecture. Unexpectedly, in a dual-species ecological model, the ezrA mutant exhibited substantially lower tolerance for H 2 O 2 and reduced competitiveness against one commensal species, Streptococcus sanguinis. Taken together, these results demonstrate that EzrA plays a key role in regulating cell division and maintaining a normal morphology in S. mutans and is required for its robust biofilm formation/interspecies competition. Therefore, EzrA protein represents a potential therapeutic target in the development of drugs controlling dental caries and other biofilm-related diseases., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Both intracellular immune sensing and extracellular innate immune sensing have been implicated in initiating macrophage proinflammatory cytokine responses to Streptococcus pneumoniae The S. pneumoniae capsule, a major virulence determinant, prevents phagocytosis, and we hypothesized that this would reduce activation of host innate inflammatory responses by preventing activation of intracellular proinflammatory signaling pathways. We investigated this hypothesis in human monocyte-derived macrophages stimulated with encapsulated or isogenic unencapsulated mutant S. pneumoniae Unexpectedly, despite strongly inhibiting bacterial internalization, the capsule resulted in enhanced inflammatory cytokine production by macrophages infected with S. pneumoniae Experiments using purified capsule material and a Streptococcus mitis mutant expressing an S. pneumoniae serotype 4 capsule indicated these differences required whole bacteria and were not due to proinflammatory effects of the capsule itself. Transcriptional profiling demonstrated relatively few differences in macrophage gene expression profiles between infections with encapsulated S. pneumoniae and those with unencapsulated S. pneumoniae , largely limited to reduced expression of proinflammatory genes in response to unencapsulated bacteria, predicted to be due to reduced activation of the NF-κB family of transcription factors. Blocking S. pneumoniae internalization using cytochalasin D had minimal effects on the inflammatory response to S. pneumoniae Experiments using murine macrophages indicated that the affected genes were dependent on Toll-like receptor 2 (TLR2) activation, although not through direct stimulation of TLR2 by capsule polysaccharide. Our data demonstrate that the early macrophage proinflammatory response to S. pneumoniae is mainly dependent on extracellular bacteria and reveal an unexpected proinflammatory effect of encapsulated S. pneumoniae that could contribute to disease pathogenesis. IMPORTANCE Multiple extra- and intracellular innate immune receptors have been identified that recognize Streptococcus pneumoniae , but the relative contributions of intra- versus extracellular bacteria to the inflammatory response were unknown. We have shown that intracellular S. pneumoniae contributes surprisingly little to the inflammatory responses, with production of important proinflammatory cytokines largely dependent on extracellular bacteria. Furthermore, although we expected the S. pneumoniae polysaccharide capsule to block activation of the host immune system by reducing bacterial internalization and therefore activation of intracellular innate immune receptors, there was an increased inflammatory response to encapsulated compared to unencapsulated bacteria, which is likely to contribute to disease pathogenesis., (Copyright © 2019 Periselneris et al.)

β-lactam antibiotics interfere with cross-linking of the bacterial cell wall, but the killing mechanism of this important class of antibiotics is not fully understood. Serendipitously we found that sub-lethal doses of β-lactams rescue growth and prevent spontaneous lysis of Staphylococcus aureus mutants lacking the widely conserved chaperone ClpX, and we reasoned that a better understanding of the clpX phenotypes could provide novel insights into the downstream effects of β-lactam binding to the PBP targets. Super-resolution imaging revealed that clpX cells display aberrant septum synthesis, and initiate daughter cell separation prior to septum completion at 30°C, but not at 37°C, demonstrating that ClpX becomes critical for coordinating the S. aureus cell cycle as the temperature decreases. FtsZ localization and dynamics were not affected in the absence of ClpX, suggesting that ClpX affects septum formation and autolytic activation downstream of Z-ring formation. Interestingly, oxacillin antagonized the septum progression defects of clpX cells and prevented lysis of prematurely splitting clpX cells. Strikingly, inhibitors of wall teichoic acid (WTA) biosynthesis that work synergistically with β-lactams to kill MRSA synthesis also rescued growth of the clpX mutant, as did genetic inactivation of the gene encoding the septal autolysin, Sle1. Taken together, our data support a model in which Sle1 causes premature splitting and lysis of clpX daughter cells unless Sle1-dependent lysis is antagonized by β-lactams or by inhibiting an early step in WTA biosynthesis. The finding that β-lactams and inhibitors of WTA biosynthesis specifically prevent lysis of a mutant with dysregulated autolytic activity lends support to the idea that PBPs and WTA biosynthesis play an important role in coordinating cell division with autolytic splitting of daughter cells, and that β-lactams do not kill S. aureus simply by weakening the cell wall., Competing Interests: The authors have declared that no competing interests exist.

Bacterial type IV secretion systems (T4SS) are a highly diversified but evolutionarily related family of macromolecule transporters that can secrete proteins and DNA into the extracellular medium or into target cells. It was recently shown that a subtype of T4SS harboured by the plant pathogen Xanthomonas citri transfers toxins into target cells. Here, we show that a similar T4SS from the multi-drug-resistant opportunistic pathogen Stenotrophomonas maltophilia is proficient in killing competitor bacterial species. T4SS-dependent duelling between S. maltophilia and X. citri was observed by time-lapse fluorescence microscopy. A bioinformatic search of the S. maltophilia K279a genome for proteins containing a C-terminal domain conserved in X. citri T4SS effectors (XVIPCD) identified twelve putative effectors and their cognate immunity proteins. We selected a putative S. maltophilia effector with unknown function (Smlt3024) for further characterization and confirmed that it is indeed secreted in a T4SS-dependent manner. Expression of Smlt3024 in the periplasm of E. coli or its contact-dependent delivery via T4SS into E. coli by X. citri resulted in reduced growth rates, which could be counteracted by expression of its cognate inhibitor Smlt3025 in the target cell. Furthermore, expression of the VirD4 coupling protein of X. citri can restore the function of S. maltophilia ΔvirD4, demonstrating that effectors from one species can be recognized for transfer by T4SSs from another species. Interestingly, Smlt3024 is homologous to the N-terminal domain of large Ca2+-binding RTX proteins and the crystal structure of Smlt3025 revealed a topology similar to the iron-regulated protein FrpD from Neisseria meningitidis which has been shown to interact with the RTX protein FrpC. This work expands our current knowledge about the function of bacteria-killing T4SSs and increases the panel of effectors known to be involved in T4SS-mediated interbacterial competition, which possibly contribute to the establishment of S. maltophilia in clinical and environmental settings., Competing Interests: The authors have declared that no competing interests exist.

Purpose: Bacillus causes a sight-threating infection of the posterior segment of the eye. The robust intraocular inflammatory response in this disease is likely activated via host innate receptor interactions with components of the Bacillus cell envelope. S-layer proteins (SLPs) of some Gram-positive pathogens contribute to the pathogenesis of certain infections. The potential contributions of SLPs in eye infection pathogenesis have not been considered. Here, we explored the role of a Bacillus SLP (SlpA) in endophthalmitis pathogenesis., Methods: The phenotypes and infectivity of wild-type (WT) and S-layer deficient (ΔslpA) Bacillus thuringiensis were compared. Experimental endophthalmitis was induced in C57BL/6J mice by intravitreally injecting 100-CFU WT or ΔslpA B. thuringiensis. Infected eyes were analyzed by bacterial counts, retinal function analysis, histology, and inflammatory cell influx. SLP-induced inflammation was also analyzed in vitro. Muller cells (MIO-M1) were treated with purified SLP. Nuclear factor-κB (NF-κB) DNA binding was measured by ELISA and expression of proinflammatory mediators from Muller cells was measured by RT-qPCR., Results: Tested phenotypes of WT and ΔslpA B. thuringiensis were similar, with the exception of absence of the S-layer in the ΔslpA mutant. Intraocular growth of WT and ΔslpA B. thuringiensis was also similar. However, eyes infected with the ΔslpA mutant had significantly reduced inflammatory cell influx, less inflammatory damage to the eyes, and significant retention of retinal function compared with WT-infected eyes. SLP was also a potent stimulator of the NF-κB pathway and induced the expression of proinflammatory mediators (IL6, TNFα, CCL2, and CXCL-1) in human retinal Muller cells., Conclusions: Taken together, our results suggest that SlpA contributes to the pathogenesis of Bacillus endophthalmitis, potentially by triggering innate inflammatory pathways in the retina.

Innate immunity is regulated by phagocytic cells and is critical for host control of bacterial infection. In many bacteria, the type VI secretion system (T6SS) can affect bacterial virulence in certain environments, but little is known about the mechanisms underlying T6SS regulation of innate immune responses during infection in vivo. Here, we developed an infection model by microinjecting bacteria into the tail vein muscle of 3-day-post-fertilized zebrafish larvae, and found that both macrophages and neutrophils are essential for bacterial clearance. Further study revealed that EvpP plays a critical role in promoting the pathogenesis of Edwardsiella piscicida (E. piscicida) via inhibiting the phosphorylation of Jnk signaling to reduce the expression of chemokine (CXC motif) ligand 8 (cxcl8a), matrix metallopeptidase 13 (mmp13) and interleukin-1β (IL-1β) in vivo. Subsequently, by utilizing Tg (mpo:eGFP +/+ ) zebrafish larvae for E. piscicida infection, we found that the EvpP-inhibited Jnk-caspy (caspase-1 homolog) inflammasome signaling axis significantly suppressed the recruitment of neutrophils to infection sites, and the caspy- or IL-1β-morpholino (MO) knockdown larvae were more susceptible to infection and failed to restrict bacterial colonization in vivo. taken together, this interaction improves our understanding about the complex and contextual role of a bacterial T6SS effector in modulating the action of neutrophils during infection, and offers new insights into the warfare between bacterial weapons and host immunological surveillance., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Mycobacterium tuberculosis employs defense mechanisms to protect itself from reactive oxygen species (ROS)-mediated cytotoxicity inside macrophages. In the current study, we found that a secretory protein of M. tuberculosis PPE2 disrupted the assembly of NADPH oxidase complex. PPE2 inhibited NADPH oxidase-mediated ROS generation in RAW 264.7 macrophages and peritoneal macrophages from BALB/c mice. PPE2 interacted with the cytosolic subunit of NADPH oxidase, p67 phox , and prevented translocation of p67 phox and p47 phox to the membrane, resulting in decreased NADPH oxidase activity. Trp236 residue present in the SH3-like domain of PPE2 was found to be critical for its interaction with p67 phox Trp236Ala mutant of PPE2 did not interact with p67 phox and thereby did not affect ROS generation. M. tuberculosis expressing PPE2 and PPE2-null mutants complemented with PPE2 survived better than PPE2-null mutants in infected RAW 264.7 macrophages. Altogether, this study suggests that PPE2 inhibits NADPH oxidase-mediated ROS production to favor M. tuberculosis survival in macrophages. The findings that M. tuberculosis PPE2 protein is involved in the modulation of oxidative response in macrophages will help us in improving our knowledge of host-pathogen interactions and the application of better therapeutics against tuberculosis., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Thermostability plays an important role in the application of L-asparaginase in the pharmaceutical and food industries. Understanding the key residues and structures that influence thermostability in L-asparaginase is necessary to obtain suitable L-asparaginase candidates. In this study, special residues and structures that altered thermostability in thermophilic L-asparaginase and non-thermophilic L-asparaginase II were identified. Interchanging these special residues and structures of L-asparaginases from the four strains, that is, Pyrococcus yayanosii CH1 (PYA), Thermococcus gammatolerans (TGA), Bacillus subtilis (BSA II), and Escherichia coli (ECA II), revealed the 51st and 298th residues of PYA (corresponding to 57th, 305th residues of ECA II) as the key residues responsible for thermal stability of thermophilic L-asparaginase and non-thermophilic L-asparaginase II. Moreover, the C terminal tightness, loop rigidity, and low surface charge around activity sites were of great significance to the thermostability of L-asparaginase. This study therefore revealed the crucial amino acid residues and structures responsible for the difference in thermostability of the thermophilic and non-thermophilic L-asparaginase and provides a reference for engineering thermostability in L-asparaginase II.

The SaeRS two-component system in Staphylococcus aureus is critical for regulation of many virulence genes, including hla , which encodes alpha-toxin. However, the impact of regulation of alpha-toxin by Sae on S. aureus pathogenesis has not been directly addressed. Here, we mutated the SaeR-binding sequences in the hla regulatory region and determined the contribution of this mutation to hla expression and pathogenesis in strain USA300 JE2. Western blot analyses revealed drastic reduction of alpha-toxin levels in the culture supernatants of SaeR-binding mutant in contrast to the marked alpha-toxin production in the wild type. The SaeR-binding mutation had no significant effect on alpha-toxin regulation by Agr, MgrA, and CcpA. In animal studies, we found that the SaeR-binding mutation did not contribute to USA300 JE2 pathogenesis using a rat infective endocarditis model. However, in a rat skin and soft tissue infection model, the abscesses on rats infected with the mutant were significantly smaller than the abscesses on those infected with the wild type but similar to the abscesses on those infected with a saeR mutant. These studies indicated that there is a direct effect of hla regulation by SaeR on pathogenesis but that the effect depends on the animal model used., (Copyright © 2019 American Society for Microbiology.)

Helicobacter pylori is a pathogen that chronically colonizes the stomachs of approximately half of the world's population and contributes to the development of gastric inflammation. We demonstrated previously in vivo that H. pylori uses motility to preferentially colonize injury sites in the mouse stomach. However, the chemoreceptor responsible for sensing gastric injury has not yet been identified. In this study, we utilized murine gastric organoids (gastroids) and mutant H. pylori strains to investigate the components necessary for H. pylori chemotaxis. High-intensity 730-nm light (two-photon photodamage) was used to cause single-cell damage in gastroids, and repair of the damage was monitored over time; complete repair occurred within ∼10 min in uninfected gastroids. Wild-type H. pylori accumulated at the damage site after gastric damage induction. In contrast, mutants lacking motility (Δ motB ) or chemotaxis (Δ cheY ) did not accumulate at the injury site. Using mutants lacking individual chemoreceptors, we found that only TlpB was required for H. pylori accumulation, while TlpA, TlpC, and TlpD were dispensable. All strains that were able to accumulate at the damage site limited repair. When urea (an identified chemoattractant sensed by TlpB) was microinjected into the gastroid lumen, it prevented the accumulation of H. pylori at damage sites. Overall, our findings demonstrate that H. pylori colonizes and limits repair at damage sites via chemotactic motility that requires the TlpB chemoreceptor to sense signals generated by gastric epithelial cells., (Copyright © 2019 American Society for Microbiology.)

Stenotrophomonas maltophilia is an emerging opportunistic and nosocomial pathogen. S. maltophilia is also a risk factor for lung exacerbations in cystic fibrosis patients. S. maltophilia attaches to various mammalian cells, and we recently documented that the bacterium encodes a type II secretion system which triggers detachment-induced apoptosis in lung epithelial cells. We have now confirmed that S. maltophilia also encodes a type IVA secretion system (VirB/VirD4 [VirB/D4] T4SS) that is highly conserved among S. maltophilia strains and, looking beyond the Stenotrophomonas genus, is most similar to the T4SS of Xanthomonas To define the role(s) of this T4SS, we constructed a mutant of strain K279a that is devoid of secretion activity due to loss of the VirB10 component. The mutant induced a higher level of apoptosis upon infection of human lung epithelial cells, indicating that a T4SS effector(s) has antiapoptotic activity. However, when we infected human macrophages, the mutant triggered a lower level of apoptosis, implying that the T4SS also elaborates a proapoptotic factor(s). Moreover, when we cocultured K279a with strains of Pseudomonas aeruginosa , the T4SS promoted the growth of S. maltophilia and reduced the numbers of heterologous bacteria, signaling that another effector(s) has antibacterial activity. In all cases, the effect of the T4SS required S. maltophilia contact with its target. Thus, S. maltophilia VirB/D4 T4SS appears to secrete multiple effectors capable of modulating death pathways. That a T4SS can have anti- and prokilling effects on different targets, including both human and bacterial cells, has, to our knowledge, not been seen before., (Copyright © 2019 American Society for Microbiology.)

Salmonella enterica serovar Typhimurium ( S. Typhimurium) induces inflammatory changes in the ceca of streptomycin-pretreated mice. In this mouse model of colitis, the type III secretion system 1 (T3SS-1) has been shown to induce rapid inflammatory change in the cecum at early points, 10 to 24 h after infection. Five proteins, SipA, SopA, SopB, SopD, and SopE2, have been identified as effectors involved in eliciting intestinal inflammation within this time range. In contrast, a T3SS-1-deficient strain was shown to exhibit inflammatory changes in the cecum at 72 to 120 h postinfection. However, the effectors eliciting T3SS-1-independent inflammation remain to be clarified. In this study, we focused on two T3SS-2 phenotypes, macrophage proliferation and cytotoxicity, to identify the T3SS-2 effectors involved in T3SS-1-independent inflammation. We identified a mutant strain that could not induce cytotoxicity in a macrophage-like cell line and that reduced intestinal inflammation in streptomycin-pretreated mice. We also identified five T3SS-2 effectors, SifA, SpvB, SseF, SseJ, and SteA, associated with T3SS-1-independent macrophage cytotoxicity. We then constructed a strain lacking T3SS-1 and all the five T3SS-2 effectors, termed T1S5. The S. Typhimurium T1S5 strain significantly reduced cytotoxicity in macrophages in the same manner as a mutant invA spiB strain (T1T2). Finally, the T1S5 strain elicited no inflammatory changes in the ceca of streptomycin-pretreated mice. We conclude that these five T3SS-2 effectors contribute to T3SS-1-independent inflammation., (Copyright © 2019 American Society for Microbiology.)

DNA methyltransferases are ubiquitous enzymes conserved in bacteria, plants and opisthokonta. These enzymes, which methylate cytosines, are involved in numerous biological processes, notably development. In mammals and higher plants, methylation patterns established and maintained by the cytosine DNA methyltransferases (DMTs) are essential to zygotic development. In fungi, some members of an extensively conserved fungal-specific DNA methyltransferase class are both mediators of the Repeat Induced Point mutation (RIP) genome defense system and key players of sexual reproduction. Yet, no DNA methyltransferase activity of these purified RID (RIP deficient) proteins could be detected in vitro. These observations led us to explore how RID-like DNA methyltransferase encoding genes would play a role during sexual development of fungi showing very little genomic DNA methylation, if any. To do so, we used the model ascomycete fungus Podospora anserina. We identified the PaRid gene, encoding a RID-like DNA methyltransferase and constructed knocked-out ΔPaRid defective mutants. Crosses involving P. anserina ΔPaRid mutants are sterile. Our results show that, although gametes are readily formed and fertilization occurs in a ΔPaRid background, sexual development is blocked just before the individualization of the dikaryotic cells leading to meiocytes. Complementation of ΔPaRid mutants with ectopic alleles of PaRid, including GFP-tagged, point-mutated and chimeric alleles, demonstrated that the catalytic motif of the putative PaRid methyltransferase is essential to ensure proper sexual development and that the expression of PaRid is spatially and temporally restricted. A transcriptomic analysis performed on mutant crosses revealed an overlap of the PaRid-controlled genetic network with the well-known mating-types gene developmental pathway common to an important group of fungi, the Pezizomycotina., Competing Interests: The authors have declared that no competing interests exist.

A comparative proteomic analysis was utilized to evaluate similarities and differences in membrane samples derived from the cariogenic bacterium Streptococcus mutans, including the wild-type strain and four mutants devoid of protein translocation machinery components, specifically ∆ffh, ∆yidC1, ∆yidC2, or ∆ffh/yidC1. The purpose of this work was to determine the extent to which the encoded proteins operate individually or in concert with one another and to identify the potential substrates of the respective pathways. Ffh is the principal protein component of the signal recognition particle (SRP), while yidC1 and yidC2 are dual paralogs encoding members of the YidC/Oxa/Alb family of membrane-localized chaperone insertases. Our results suggest that the co-translational SRP pathway works in concert with either YidC1 or YidC2 specifically, or with no preference for paralog, in the insertion of most membrane-localized substrates. A few instances were identified in which the SRP pathway alone, or one of the YidCs alone, appeared to be most relevant. These data shed light on underlying reasons for differing phenotypic consequences of ffh, yidC1 or yidC2 deletion. Our data further suggest that many membrane proteins present in a ∆yidC2 background may be non-functional, that ∆yidC1 is better able to adapt physiologically to the loss of this paralog, that shared phenotypic properties of ∆ffh and ∆yidC2 mutants can stem from impacts on different proteins, and that independent binding to ribosomal proteins is not a primary functional activity of YidC2. Lastly, genomic mutations accumulate in a ∆yidC2 background coincident with phenotypic reversion, including an apparent W138R suppressor mutation within yidC1., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Introduction: Salmonella species are gram-negative facultative intracellular bacteria that are common causes of foodborne illness in North America. Infections by Salmonella during pregnancy are a significant cause of fetal loss in domestic livestock, and fetal and maternal mortality in mice. Furthermore, Salmonella infection is associated with miscarriage, stillbirth and preterm birth in pregnant women. Despite these collective associations, the extent to which Salmonella can infect the human placenta has not been investigated., Methods: Human placental villous explants from several gestational ages were exposed to Salmonella enterica serovar Typhimurium (STm) ex vivo. Infection was assessed by colony forming unit assay and whole mount immunofluorescence (WMIF)., Results: Viable bacteria were recovered from placental villous explants of all gestational ages tested, but the bacterial burden was highest in 1st trimester explants. Bacterial numbers did not change appreciably with time post-infection in explants from any gestational age examined, suggesting that STm does not proliferate in placental villi. Exposure of villous explants to STm strains defective for the type III secretion systems revealed that Salmonella pathogenicity island 1 is essential for optimal invasion. In contrast to placental explants, STm infected and proliferated within villous cytotrophoblast cells isolated from term placentas. WMIF demonstrated that STm was restricted primarily to the syncytiotrophoblast layer in infected placentas., Discussion: Our study demonstrates that STm can invade into the syncytiotrophoblast but does not subsequently proliferate. Thus, the syncytiotrophoblast may function as a barrier to STm infection of the fetus., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Drug resistance bacteria produce various proteins to combat xenobiotic, physical, and environmental stresses. These proteins play a pivotal role in bacterial cell survival, and thus are the focus of extensive research as potential targets for anti-bacterial drug discovery. During the current study, the MRSA252 (Methicillin-resistant Staphylococcus aureus) anti-σ B factor antagonist (RsbV) protein was expressed to study its structure, and function in vitro. RsbV and anti-σ B factor (RsbW) are involved in the transcription of σ B factor under stress conditions to help bacterial cell survival. The production of recombinant RsbV protein is important for ligand binding studies, and for new inhibitor identification. In order to have sufficient yield of RsbV protein for structural and functional analysis, the MRSA252 rsbV:pET25b clone was transformed into the Escherichia coli BL21(DE3). Purified RsbV protein was analyzed for structure and functions by using circular dichroism (CD) spectroscopy, and reactive oxygen species (ROS) activity assay, respectively. The CD analysis demonstrated that MRSA252 RsbV has a globular nature with α-helices, and β-strands. It is stable above the ambient temperature. RsbV protein in a cell-based antioxidant assay showed >20-fold more potent inhibition, as compared to drug ibuprofen. The structural element and functional analysis of RsbV protein indicated that RsbV protein is produced in MRSA252 probably as an active regulatory factor under physical, and chemical stresses., (Copyright © 2019. Published by Elsevier B.V.)

KatB, a salt-inducible Mn-catalase, protects the cyanobacterium Anabaena from salinity/oxidative stress. In this report, we provide distinctive insights into the biological-biochemical function of KatB at the molecular level. Anabaena overexpressing the wild-type KatB protein (KatBWT) detoxified H 2 O 2 efficiently, showing reduced burden of reactive oxygen species compared with the strain overproducing KatBF2V (wherein F-2 is replaced by V). Correspondingly, the KatBWT protein also displayed several folds more activity than KatBF2V. Interestingly, the KatB variants with large hydrophobic amino acids (F/W/Y) were more compact, showed enhanced activity, and were resistant to thermal/chemical denaturation than variants with smaller residues (G/A/V) at the second position. X-ray crystallography-based analysis showed that F-2 was required for appropriate interactions between two subunits. These contacts provided stability to the hexamer, making it more compact. F-2, through its interaction with F-66 and W-43, formed the proper hydrophobic pocket that held the active site together. Consequently, only residues that supported activity (i.e., F/Y/W) were selected at the second position in Mn-catalases during evolution. This study (a) demonstrates that modification of nonactive site residues can alter the response of catalases to environmental stress and (b) has expanded the scope of amino acids that can be targeted for rational protein engineering in plants., (© 2019 John Wiley & Sons Ltd.)

The circadian clock controls 24-h biological rhythms in our body, influencing many time-related activities such as sleep and wake. The simplest circadian clock is found in cyanobacteria, with the proteins KaiA, KaiB, and KaiC generating a self-sustained circadian oscillation of KaiC phosphorylation and dephosphorylation. KaiA activates KaiC phosphorylation by binding the A-loop of KaiC, while KaiB attenuates the phosphorylation by sequestering KaiA from the A-loop. Structural analysis revealed that magnesium regulates the phosphorylation and dephosphorylation of KaiC by dissociating from and associating with catalytic Glu residues that activate phosphorylation and dephosphorylation, respectively. High magnesium causes KaiC to dephosphorylate, whereas low magnesium causes KaiC to phosphorylate. KaiC alone behaves as an hourglass timekeeper when the magnesium concentration is alternated between low and high levels in vitro. We suggest that a magnesium-based hourglass timekeeping system may have been used by ancient cyanobacteria before magnesium homeostasis was established.

Some methylmercury (MeHg) is converted to inorganic mercury (Hg2+) after incorporation into human and animal tissues, where it can remain for a long time. To determine the overall toxicity of MeHg in tissues, studies should evaluate low concentrations of Hg2+. Although demethylation is involved, the participating enzymes or underlying mechanisms are unknown; in addition, the low cell membrane permeability of Hg2+ makes these analyses challenging. We established model cell lines to assess toxicities of low concentrations of Hg2+ using bacterial organomercury lyase (MerB). We engineered MerB-expressing HEK293 and HeLa cell lines that catalyze MeHg demethylation. These cells were significantly more sensitive to MeHg exposure compared to the parental cells. MeHg treatment remarkably induced metallothioneins (MTs) and hemeoxygenase-1 (HMOX-1) mRNAs and modest expression of superoxide dismutase 1, whereas catalase and glutathione peroxidase 1 mRNAs were not up-regulated. merB knockdown using small interfering RNA supported the induction of MT and HMOX-1 mRNA by MerB enzymatic activity. Pretreatment with Trolox, a water-soluble vitamin E analog, did not inhibit MeHg-induced elevation of MT-Ix and HMOX-1 mRNAs in MerB-expressing cells, suggesting that Hg2+ works independently of reactive oxygen species generation. Similar results were obtained in cells expressing MerB, suggesting that high MTs and HMOX-1 induction and cytotoxicity are common cellular responses to low intracellular Hg2+ concentrations. This is the first study to establish cell lines that demethylate intracellular MeHg to Hg2+ using bacterial MerB for overcoming the low membrane permeability of Hg2+ and exploring the intracellular responses and toxicities of low Hg2+ concentrations., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The RecX protein has attracted considerable interest because the recX mutants exhibit multiple phenotypes associated with RecA functions. To further our understanding of the functional relationship between recA and recX, the effect of different stress treatments on their expression profiles, cell yield and viability were investigated. A significant correlation was found between the expression of Mycobacterium smegmatis recA and recX genes at different stages of growth, and in response to different stress treatments albeit recX exhibiting lower transcript and protein abundance at the mid-log and stationary phases of the bacterial growth cycle. To ascertain their roles in vivo, a targeted deletion of the recX and recArecX was performed in M. smegmatis. The growth kinetics of these mutant strains and their sensitivity patterns to different stress treatments were assessed relative to the wild-type strain. The deletion of recA affected normal cell growth and survival, while recX deletion showed no significant effect. Interestingly, deletion of both recX and recA genes results in a phenotype that is intermediate between the phenotypes of the ΔrecA mutant and the wild-type strain. Collectively, these results reveal a previously unrecognized role for M. smegmatis recX and support the notion that it may regulate a subset of the yet unknown genes involved in normal cell growth and DNA-damage repair.

Streptococcus pneumoniae (pneumococci) is a leading cause of severe bacterial meningitis in many countries worldwide. To characterize the repertoire of fitness and virulence factors predominantly expressed during meningitis we performed niche-specific analysis of the in vivo proteome in a mouse meningitis model, in which bacteria are directly inoculated into the cerebrospinal fluid (CSF) cisterna magna. We generated a comprehensive mass spectrometry (MS) spectra library enabling bacterial proteome analysis even in the presence of eukaryotic proteins. We recovered 200,000 pneumococci from CSF obtained from meningitis mice and by MS we identified 685 pneumococci proteins in samples from in vitro filter controls and 249 in CSF isolates. Strikingly, the regulatory two-component system ComDE and substrate-binding protein AliB of the oligopeptide transporter system were exclusively detected in pneumococci recovered from the CSF. In the mouse meningitis model, AliB-, ComDE-, or AliB-ComDE-deficiency resulted in attenuated meningeal inflammation and disease severity when compared to wild-type pneumococci indicating the crucial role of ComDE and AliB in pneumococcal meningitis. In conclusion, we show here mechanisms of pneumococcal adaptation to a defined host compartment by a proteome-based approach. Further, this study provides the basis of a promising strategy for the identification of protein antigens critical for invasive disease caused by pneumococci and other meningeal pathogens., Competing Interests: The authors have declared that no competing interests exist.

The pathogen Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, enacts unique transcriptional regulatory mechanisms when subjected to host-derived stresses. Initiation of transcription by the Mycobacterial RNA polymerase (RNAP) has previously been shown to exhibit different open complex kinetics and stabilities relative to Escherichia coli (Eco) RNAP. However, transcription initiation rates also depend on the kinetics following open complex formation such as initial nucleotide incorporation and subsequent promoter escape. Here, using a real-time fluorescence assay, we present the first in-depth kinetic analysis of initial transcription and promoter escape for the Mtb RNAP. We show that in relation to Eco RNAP, Mtb displays slower initial nucleotide incorporation but faster overall promoter escape kinetics on the Mtb rrnAP3 promoter. Furthermore, in the context of the essential transcription factors CarD and RbpA, Mtb promoter escape is slowed via differential effects on initially transcribing complexes. Finally, based on their ability to increase the rate of open complex formation and decrease the rate of promoter escape, we suggest that CarD and RbpA are capable of activation or repression depending on the rate-limiting step of a given promoter's basal initiation kinetics., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Staphylococcus aureus infection relies on iron acquisition from its host. S. aureus takes up iron through heme uptake by the iron-responsive surface determinant (Isd) system and by the production of iron-scavenging siderophores. Staphyloferrin B (SB) is a siderophore produced by the 9-gene sbn gene cluster for SB biosynthesis and efflux. Recently, the ninth gene product, SbnI, was determined to be a free l-serine kinase that produces O- phospho-l-serine (OPS), a substrate for SB biosynthesis. Previous studies have also characterized SbnI as a DNA-binding regulatory protein that senses heme to control sbn gene expression for SB synthesis. Here, we present crystal structures at 1.9-2.1 Å resolution of a SbnI homolog from Staphylococcus pseudintermedius (SpSbnI) in both apo form and in complex with ADP, a product of the kinase reaction; the latter confirmed the active-site location. The structures revealed that SpSbnI forms a dimer through C-terminal domain swapping and a dimer of dimers through intermolecular disulfide formation. Heme binding had only a modest effect on SbnI enzymatic activity, suggesting that its two functions are independent and structurally distinct. We identified a heme-binding site and observed catalytic heme transfer between a heme-degrading protein of the Isd system, IsdI, and SbnI. These findings support the notion that SbnI has a bifunctional role contributing precursor OPS to SB synthesis and directly sensing heme to control expression of the sbn locus. We propose that heme transfer from IsdI to SbnI enables S. aureus to control iron source preference according to the sources available in the environment., (© 2019 Verstraete et al.)

Clostridium difficile is the leading cause of antibiotic-associated diarrhea in adults. During infection, C. difficile must detect the host environment and induce an appropriate survival strategy. Signal transduction networks involving serine/threonine kinases (STKs) play key roles in adaptation, as they regulate numerous physiological processes. PrkC of C. difficile is an STK with two PASTA domains. We showed that PrkC is membrane associated and is found at the septum. We observed that deletion of prkC affects cell morphology with an increase in mean size, cell length heterogeneity, and presence of abnormal septa. A Δ prkC mutant was able to sporulate and germinate but was less motile and formed more biofilm than the wild-type strain. Moreover, a Δ prkC mutant was more sensitive to antimicrobial compounds that target the cell envelope, such as the secondary bile salt deoxycholate, cephalosporins, cationic antimicrobial peptides, and lysozyme. This increased susceptibility was not associated with differences in peptidoglycan or polysaccharide II composition. However, the Δ prkC mutant had less peptidoglycan and released more polysaccharide II into the supernatant. A proteomic analysis showed that the majority of C. difficile proteins associated with the cell wall were less abundant in the Δ prkC mutant than the wild-type strain. Finally, in a hamster model of infection, the Δ prkC mutant had a colonization delay that did not significantly affect overall virulence., (Copyright © 2019 American Society for Microbiology.)

Clostridioides difficile toxin B (TcdB) is an intracellular toxin responsible for many of the pathologies of C. difficile infection. The two variant forms of TcdB (TcdB1 and TcdB2) share 92% sequence identity but have reported differences in rates of cell entry, autoprocessing, and overall toxicity. This 2,366-amino-acid, multidomain bacterial toxin glucosylates and inactivates small GTPases in the cytosol of target cells, ultimately leading to cell death. Successful cell entry and intoxication by TcdB are known to involve various conformational changes in the protein, including a proteolytic autoprocessing event. Previous studies found that amino acids 1753 to 1852 influence the conformational states of the proximal carboxy-terminal domain of TcdB and could contribute to differences between TcdB1 and TcdB2. In the current study, a combination of approaches was used to identify sequences within the region from amino acids 1753 to 1852 that influence the conformational integrity and cytotoxicity of TcdB2. Four deletion mutants with reduced cytotoxicity were identified, while one mutant, TcdB2 Δ1769-1787 , exhibited no detectable cytotoxicity. TcdB2 Δ1769-1787 underwent spontaneous autoprocessing and was unable to interact with CHO-K1 or HeLa cells, suggesting a potential change in the conformation of the mutant protein. Despite the putative alteration in structural stability, vaccination with TcdB2 Δ1769-1787 induced a TcdB2-neutralizing antibody response and protected against C. difficile disease in a mouse model. These findings indicate that the 19-amino-acid region spanning residues 1769 to 1787 in TcdB2 is crucial to cytotoxicity and the structural regulation of autoprocessing and that TcdB2 Δ1769-1787 is a promising candidate for vaccination., (Copyright © 2019 American Society for Microbiology.)

Enclosure experiments are frequently used to investigate the impact of changing environmental conditions on microbial assemblages. Yet, how the incubation itself challenges complex bacterial communities is thus far unknown. In this study, metaproteomic profiling, 16S rRNA gene analyses, and cell counts were combined to evaluate bacterial communities derived from marine, mesohaline, and oligohaline conditions after long-term batch incubations. Early in the experiment, the three bacterial communities were highly diverse and differed significantly in their compositions. Manipulation of the enclosures with terrigenous dissolved organic carbon resulted in notable differences compared to the control enclosures at this early phase of the experiment. However, after 55 days, bacterial communities in the manipulated and the control enclosures under marine and mesohaline conditions were all dominated by gammaproteobacterium Spongiibacter In the oligohaline enclosures, actinobacterial cluster I of the hgc group (hgc-I) remained abundant in the late phase of the incubation. Metaproteome analyses suggested that the ability to use outer membrane-based internal energy stores, in addition to the previously described grazing resistance, may enable the gammaproteobacterium Spongiibacter to prevail in long-time incubations. Under oligohaline conditions, the utilization of external recalcitrant carbon appeared to be more important (hgc-I). Enclosure experiments with complex natural microbial communities are important tools to investigate the effects of manipulations. However, species-specific properties, such as individual carbon storage strategies, can cause manipulation-independent effects and need to be considered when interpreting results from enclosures. IMPORTANCE In microbial ecology, enclosure studies are often used to investigate the effect of single environmental factors on complex bacterial communities. However, in addition to the manipulation, unintended effects ("bottle effect") may occur due to the enclosure itself. In this study, we analyzed the bacterial communities that originated from three different salinities of the Baltic Sea, comparing their compositions and physiological activities both at the early stage and after 55 days of incubation. Our results suggested that internal carbon storage strategies impact the success of certain bacterial species, independent of the experimental manipulation. Thus, while enclosure experiments remain valid tools in environmental research, microbial community composition shifts must be critically followed. This investigation of the metaproteome during long-term batch enclosures expanded our current understanding of the so-called "bottle effect," which is well known to occur during enclosure experiments., (Copyright © 2019 American Society for Microbiology.)

The bacterial flagellar motor is a molecular machine that can rotate the flagellar filament at high speed. The rotation is generated by the stator-rotor interaction, coupled with an ion flux through the torque-generating stator. Here we employed cryo-electron tomography to visualize the intact flagellar motor in the Lyme disease spirochete, Borrelia burgdorferi . By analyzing the motor structures of wild-type and stator-deletion mutants, we not only localized the stator complex in situ, but also revealed the stator-rotor interaction at an unprecedented detail. Importantly, the stator-rotor interaction induces a conformational change in the flagella C-ring. Given our observation that a non-motile mutant, in which proton flux is blocked, cannot generate the similar conformational change, we propose that the proton-driven torque is responsible for the conformational change required for flagellar rotation., Competing Interests: YC, KM, XZ, SN, MM, JL No competing interests declared, (© 2019, Chang et al.)

Phosphorus acquisition is critical for life. In low phosphate conditions, some species of bacteria have evolved mechanisms to import reduced phosphorus compounds, such as phosphite and hypophosphite, as alternative phosphorus sources. Uptake is facilitated by high-affinity periplasmic binding proteins (PBPs) that bind cargo in the periplasm and shuttle it to an ATP-binding cassette (ABC)-transporter in the bacterial inner membrane. PtxB and HtxB are the PBPs responsible for binding phosphite and hypophosphite, respectively. They recognize the P-H bond of phosphite/hypophosphite via a conserved P-H...π interaction, which confers nanomolar dissociation constants for their respective ligands. PtxB also has a low-level binding affinity for phosphate and hypophosphite, whilst HtxB can facilitate phosphite uptake in vivo. However, HtxB does not bind phosphate, thus the HtxBCDE transporter has recently been successfully exploited for biocontainment of genetically modified organisms by phosphite-dependent growth. Here we use a combination of X-ray crystallography, NMR and Microscale Thermophoresis to show that phosphite binding to HtxB depends on the protonation state of the ligand, suggesting that pH may effect the efficiency of phosphite uptake by HtxB in biotechnology applications.

The mycobacterial SenX3-RegX3 two-component system consists of the SenX3 sensor histidine kinase and its cognate RegX3 response regulator. This system is a phosphorelay-based regulatory system involved in sensing environmental P i levels and induction of genes required for P i acquisition under P i -limiting conditions. Here we demonstrate that overexpression of the kinase domain of Mycobacterium tuberculosis PknB (PknB-KD Mtb ) inhibits the transcriptional activity of RegX3 of both M. tuberculosis and Mycobacterium smegmatis (RegX3 Mtb and RegX3 Ms , respectively). Mass spectrometry results, along with those of in vitro phosphorylation and complementation analyses, revealed that PknB kinase activity inhibits the transcriptional activity of RegX3 Mtb through phosphorylation events at Thr-100, Thr-191, and Thr-217. Electrophoretic mobility shift assays disclosed that phosphorylation of Thr-191 and Thr-217 abolishes the DNA-binding ability of RegX3 Mtb and that Thr-100 phosphorylation likely prevents RegX3 Mtb from being activated through conformational changes induced by SenX3-mediated phosphorylation. We propose that the convergence of the PknB and SenX3-RegX3 signaling pathways might enable mycobacteria to integrate environmental P i signals with the cellular replication state to adjust gene expression in response to P i availability., (© 2019 Park et al.)

Background: Staphylococcus aureus remains a medical challenge in the treatment of bacterial infections. It has acquired resistance to commonly used antibiotics, and to those considered to be the last weapons in treating staphylococcal infections, such as vancomycin. Studies have revealed that S. aureus is capable of mounting a rapid response to antibiotics that target cell wall peptidoglycan biosynthesis, such as β-lactams and vancomycin. The two-component system VraSR has been linked to the coordination of this response. VraS is a histidine kinase that undergoes autophosphorylation in the presence of signals elicited upon cell wall damage and it then transfers its phosphoryl group to VraR. VraR is a response regulator protein that functions as a transcription factor. Phosphorylation of VraR leads to its dimerization, which is required for optimum binding to its target promoters. Two-component systems have been targeted for the development of antibacterial agents. Deletion of the vraS or vraR gene has been shown to re-sensitize S. aureus to β-lactams and vancomycin., Results: In this study, we explored perturbation of the VraR phosphorylation-induced activation as a means to inhibit the VraSR-mediated signal transduction pathway. We show that dimerization of VraR is essential for the phosphorylation-induced activation of VraR. A single point mutation in the dimerization interface of VraR, in which Met13 was replaced by Ala, led to the inability of VraR to dimerize and to bind optimally to the target promoter. The consequences of these in vitro molecular deficiencies are equally dramatic in vivo. Complementation of a vraR deletion S. aureus strain with the vraRM13Ala mutant gene failed to induce the cell wall stress response., Conclusions: This study highlights the potential of targeting the phosphorylation-induced dimerization of VraR to disrupt the S. aureus cell wall stress response and in turn to re-sensitize S. aureus to β-lactams and vancomycin.

The pathogen Staphylococcus aureus colonizes and infects a variety of different sites within the human body. To adapt to these different environments, S. aureus relies on a complex and finely tuned regulatory network. While some of these networks have been well-elucidated, the functions of more than 50% of the transcriptional regulators in S. aureus remain unexplored. Here, we assess the contribution of the LacI family of metabolic regulators to staphylococcal virulence. We found that inactivating the purine biosynthesis regulator purR resulted in a strain that was acutely virulent in bloodstream infection models in mice and in ex vivo models using primary human neutrophils. Remarkably, these enhanced pathogenic traits are independent of purine biosynthesis, as the purR mutant was still highly virulent in the presence of mutations that disrupt PurR's canonical role. Through the use of transcriptomics coupled with proteomics, we revealed that a number of virulence factors are differentially regulated in the absence of purR Indeed, we demonstrate that PurR directly binds to the promoters of genes encoding virulence factors and to master regulators of virulence. These results guided us into further ex vivo and in vivo studies, where we discovered that S. aureus toxins drive the death of human phagocytes and mice, whereas the surface adhesin FnbA contributes to the increased bacterial burden observed in the purR mutant. Thus, S. aureus repurposes a metabolic regulator to directly control the expression of virulence factors, and by doing so, tempers its pathogenesis., Competing Interests: Conflict of interest statement: V.J.T. is an inventor on patents and patent applications filed by New York University which are currently under commercial license to Janssen Biotech, Inc.

The ability to regulate gene expression through transcription initiation underlies the adaptability and survival of all bacteria. Recent work has revealed that the transcription machinery in many bacteria diverges from the paradigm that has been established in Escherichia coli Mycobacterium tuberculosis ( Mtb ) encodes the RNA polymerase (RNAP)-binding protein CarD, which is absent in E. coli but is required to form stable RNAP-promoter open complexes (RP o ) and is essential for viability in Mtb The stabilization of RP o by CarD has been proposed to result in activation of gene expression; however, CarD has only been examined on limited promoters that do not represent the typical promoter structure in Mtb In this study, we investigate the outcome of CarD activity on gene expression from Mtb promoters genome-wide by performing RNA sequencing on a panel of mutants that differentially affect CarD's ability to stabilize RP o In all CarD mutants, the majority of Mtb protein encoding transcripts were differentially expressed, demonstrating that CarD had a global effect on gene expression. Contrary to the expected role of CarD as a transcriptional activator, mutation of CarD led to both up- and down-regulation of gene expression, suggesting that CarD can also act as a transcriptional repressor. Furthermore, we present evidence that stabilization of RP o by CarD could lead to transcriptional repression by inhibiting promoter escape, and the outcome of CarD activity is dependent on the intrinsic kinetic properties of a given promoter region. Collectively, our data support CarD's genome-wide role of regulating diverse transcription outcomes., Competing Interests: The authors declare no conflict of interest.

Objectives: Carbapenem resistance in Pseudomonas aeruginosa is growing and results from variable mechanisms. The objectives of the current study were to investigate mechanisms of carbapenem resistance and genetic relatedness of P. aeruginosa isolates recovered in Dubai hospitals., Methods: From June 2015 through June 2016, carbapenem-nonsusceptible P. aeruginosa were collected from 4 hospitals in Dubai, and subjected to antimicrobial susceptibility testing, molecular investigation of carbapenemases by PCR-sequencing, analysis of outer membrane porin OprD2 and multidrug efflux channel MexAB-OprM levels by qPCR, and fingerprinting by ERIC-PCR., Results: Out of 1969 P. aeruginosa isolated during the study period, 471 (23.9%) showed reduced carbapenem susceptibility. Of these, 37 were analyzed and 32% of them produced VIM-type metallo-β-lactamases, including VIM-2, VIM-30, VIM-31, and VIM-42, while GES-5 and GES-9 co-existed with VIM in 5.4% of isolates. Outer membrane impermeability was observed in 73% of isolates and 75.6% displayed overproduced MexAB-OprM. ERIC-PCR revealed one large clone including most carbapenemase-producing isolates indicating clonal dissemination., Conclusion: This is the first study on carbapenem-nonsusceptible P. aeruginosa from Dubai, incriminating VIM production as well as outer membrane permeability and efflux systems as resistance mechanisms. Further studies on carbapenem-nonsusceptible P. aeruginosa in Dubai are warranted for containment of such health hazard., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Beta haemolytic Group A streptococcus (GAS) or Streptococcus pyogenes are strict human pathogens responsible for mild to severe fatal invasive infections. Even with enormous number of reports exploring the role of S. pyogenes exotoxins in its pathogenesis, inadequate knowledge on the biofilm process and the potential role of exotoxins in bacterial dissemination from matured biofilms has been a hindrance in development of effective and targeted treatments. Therefore, the present study was aimed in investigating the uncharted role of these exotoxins in biofilm process. Through our study the putative role of ciaRH in the SpeA dependent ablation of biofilm formation could be speculated and thus helping in bacterial dissemination. The seed-dispersal effect of SpeA was time and concentration dependent and seen to be consistent within various streptococcal species. Transcriptome analysis of SpeA treated S. pyogenes biofilms revealed the involvement of many transcriptional regulators (ciaRH) and response genes (luxS, shr, shp, SPy_0572), hinting towards specific mechanisms underlying the dispersal effect by SpeA. This finding opens up a discussion towards understanding a new mechanism involved in the pathogenesis of Streptococcus pyogenes and might help in understanding the bacterial infections in a better way.

Clostridium difficile infection (CDI) is a major nosocomial disease associated with significant morbidity and mortality. The pathology of CDI stems primarily from the 2 C. difficile-secreted exotoxins-toxin A (TcdA) and toxin B (TcdB)-that disrupt the tight junctions between epithelial cells leading to the loss of colonic epithelial barrier function. Here, we report the engineering of a series of monomeric and dimeric designed ankyrin repeat proteins (DARPins) for the neutralization of TcdB. The best dimeric DARPin, DLD-4, inhibited TcdB with a half maximal effective concentration (EC50) of 4 pM in vitro, representing an approximately 330-fold higher potency than the Food and Drug Administration (FDA)-approved anti-TcdB monoclonal antibody bezlotoxumab in the same assay. DLD-4 also protected mice from a toxin challenge in vivo. Cryo-electron microscopy (cryo-EM) studies revealed that the 2 constituent DARPins of DLD-4-1.4E and U3-bind the central and C-terminal regions of the delivery domain of TcdB. Competitive enzyme-linked immunosorbent assay (ELISA) studies showed that the DARPins 1.4E and U3 interfere with the interaction between TcdB and its receptors chondroitin sulfate proteoglycan 4 (CSPG4) and frizzled class receptor 2 (FZD2), respectively. Our cryo-EM studies revealed a new conformation of TcdB (both apo- and DARPin-bound at pH 7.4) in which the combined repetitive oligopeptides (CROPS) domain points away from the delivery domain. This conformation of the CROPS domain is in stark contrast to that seen in the negative-stain electron microscopy (EM) structure of TcdA and TcdB at the same pH, in which the CROPS domain bends toward and "kisses" the delivery domain. The ultrapotent anti-TcdB molecules from this study serve as candidate starting points for CDI drug development and provide new biological tools for studying the pathogenicity of C. difficile. The structural insights regarding both the "native" conformation of TcdB and the putative sites of TcdB interaction with the FZD2 receptor, in particular, should help accelerate the development of next-generation anti-C. difficile toxin therapeutics., Competing Interests: The Texas A&M University and the University of Maryland have submitted a patent application on the anti-toxin DARPins reported in this study.

Contact-dependent growth inhibition (CDI) systems are used in bacterial competition to hinder the growth of neighboring microbes. These systems utilize a two-partner secretion mechanism to display the CdiA exoprotein at the bacterial cell surface. CdiA forms a long filamentous stalk that facilitates binding to a target cell and delivery of a C-terminal toxin (CT) domain. This CT domain is processed and delivered into the cytoplasm of a target cell upon contact. CDI systems also encode a cognate immunity protein (CdiI) that protects siblings and resistant targeted cells from intoxication by high-affinity binding to the CT. CdiA CT domains vary among strains within a species, and many alleles encode enzymatic functions that target nucleic acids. This variation is thought to help drive diversity and adaptation within a species. CdiA diversity is well studied in Escherichia coli and several other bacteria, but little is known about the extent of this diversity in Pseudomonas aeruginosa . The purpose of this review is to highlight the variability that exists in CDI systems of P. aeruginosa . We show that this diversity is apparent even among strains isolated from a single geographical region, suggesting that CDI systems play an important role in the ecology of P. aeruginosa ., (Copyright © 2019 American Society for Microbiology.)

Legionella pneumophila is ubiquitous in freshwater environments, where it replicates within unicellular protozoa. However, L. pneumophila is also an accidental human pathogen that can cause Legionnaires' disease in immunocompromised individuals by uncontrolled replication within alveolar macrophages. To replicate within eukaryotic phagocytes, L. pneumophila utilizes a Dot/Icm type IV secretion system to translocate a large arsenal of over 300 effector proteins directly into host cells. In mammals, translocated effectors contribute to innate immune restriction of L. pneumophila We found previously that the effector LegC4 is important for L. pneumophila replication within a natural host protist but is deleterious to replication in a mouse model of Legionnaires' disease. In the present study, we used cultured mouse primary macrophages to investigate how LegC4 attenuates L. pneumophila replication. We found that LegC4 enhanced restriction of L. pneumophila replication within macrophages activated with tumor necrosis factor (TNF) or interferon gamma (IFN-γ). In addition, expression of legC4 was sufficient to restrict Legionella longbeachae replication within TNF- or IFN-γ-activated macrophages. Thus, this study demonstrates that LegC4 contributes to L. pneumophila clearance from healthy hosts by potentiating cytokine-mediated host defense mechanisms. IMPORTANCE Legionella spp. are natural pathogens of protozoa and accidental pathogens of humans. Innate immunity in healthy individuals effectively controls Legionella infection due in part to rapid and robust production of proinflammatory cytokines resulting from detection of Dot/Icm-translocated substrates, including effectors. Here, we demonstrate that the effector LegC4 enhances proinflammatory host restriction of Legionella by macrophages. These data suggest that LegC4 may augment proinflammatory signaling or antimicrobial activity of macrophages, a function that has not previously been observed for another bacterial effector. Further insight into LegC4 function will likely reveal novel mechanisms to enhance immunity against pathogens., (Copyright © 2019 American Society for Microbiology.)

Shigella flexneri is an intracellular bacterial pathogen that invades epithelial cells in the colonic mucosa, leading to bloody diarrhea. A previous study showed that S. flexneri forms biofilms in the presence of bile salts, through an unknown mechanism. Here, we investigated the potential role of adhesin-like autotransporter proteins in S. flexneri biofilm formation. BLAST search analysis revealed that the S. flexneri 2457T genome harbors 4 genes, S1242 , S1289 , S2406 , and icsA , encoding adhesin-like autotransporter proteins. Deletion mutants of the S1242 , S1289 , S2406 and icsA genes were generated and tested for biofilm formation. Phenotypic analysis of the mutant strains revealed that disruption of icsA abolished bile salt-induced biofilm formation. IcsA is an outer membrane protein secreted at the bacterial pole that is required for S. flexneri actin-based motility during intracellular infection. In extracellular biofilms, IcsA was also secreted at the bacterial pole and mediated bacterial cell-cell contacts and aggregative growth in the presence of bile salts. Dissecting individual roles of bile salts showed that deoxycholate is a robust biofilm inducer compared to cholate. The release of the extracellular domain of IcsA through IcsP-mediated cleavage was greater in the presence of cholate, suggesting that the robustness of biofilm formation was inversely correlated with IcsA processing. Accordingly, deletion of icsP abrogated IcsA processing in biofilms and enhanced biofilm formation., (Copyright © 2019 American Society for Microbiology.)

In preparation for division, bacteria replicate their DNA and segregate the newly formed chromosomes. A division septum then assembles between the chromosomes, and the mother cell splits into two identical daughters due to septum degradation. A major constituent of bacterial septa and of the whole cell wall is peptidoglycan (PGN), an essential cell wall polymer, formed by glycan chains of β-(1-4)-linked- N -acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), cross-linked by short peptide stems. Depending on the amino acid located at the third position of the peptide stem, PGN is classified as either Lys-type or meso-diaminopimelic acid (DAP)-type. Hydrolytic enzymes play a crucial role in the degradation of bacterial septa to split the cell wall material shared by adjacent daughter cells to promote their separation. In mycobacteria, a key PGN hydrolase, belonging to the NlpC/P60 endopeptidase family and denoted as RipA, is responsible for the degradation of septa, as the deletion of the gene encoding for this enzyme generates abnormal bacteria with multiple septa. This review provides an update of structural and functional data highlighting the central role of RipA in mycobacterial cytokinesis and the fine regulation of its catalytic activity, which involves multiple molecular partners.

The bacterial flagellar motor powers the rotation that propels the swimming bacteria. Rotational torque is generated by harnessing the flow of ions through ion channels known as stators which couple the energy from the ion gradient across the inner membrane to rotation of the rotor. Here, we used error-prone PCR to introduce single point mutations into the sodium-powered Vibrio alginolyticus/Escherichia coli chimeric stator PotB and selected for motors that exhibited motility in the presence of the sodium-channel inhibitor phenamil. We found single mutations that enable motility under phenamil occurred at two sites: (i) the transmembrane domain of PotB, corresponding to the TM region of the PomB stator from V. alginolyticus and (ii) near the peptidoglycan binding region that corresponds to the C-terminal region of the MotB stator from E. coli. Single cell rotation assays confirmed that individual flagellar motors could rotate in up to 100 µM phenamil. Using phylogenetic logistic regression, we found correlation between natural residue variation and ion source at positions corresponding to PotB F22Y, but not at other sites. Our results demonstrate that it is not only the pore region of the stator that moderates motility in the presence of ion-channel blockers., (© 2019 John Wiley & Sons Ltd.)

Several pathogenic bacteria utilize sialic acid, including host-derived N-acetylneuraminic acid (Neu5Ac), in at least two ways: they use it as a nutrient source and as a host-evasion strategy by coating themselves with Neu5Ac. Given the significant role of sialic acid in pathogenesis and host-gut colonization by various pathogenic bacteria, including Neisseria meningitidis, Haemophilus influenzae, Pasteurella multocida and Vibrio cholerae, several enzymes of the sialic acid catabolic, biosynthetic and incorporation pathways are considered to be potential drug targets. In this work, findings on the structural and functional characterization of CMP-N-acetylneuraminate synthetase (CMAS), a key enzyme in the incorporation pathway, from Vibrio cholerae are reported. CMAS catalyzes the synthesis of CMP-sialic acid by utilizing CTP and sialic acid. Crystal structures of the apo and the CDP-bound forms of the enzyme were determined, which allowed the identification of the metal cofactor Mg 2+ in the active site interacting with CDP and the invariant Asp215 residue. While open and closed structural forms of the enzyme from eukaryotic and other bacterial species have already been characterized, a partially closed structure of V. cholerae CMAS (VcCMAS) observed upon CDP binding, representing an intermediate state, is reported here. The kinetic data suggest that VcCMAS is capable of activating the two most common sialic acid derivatives, Neu5Ac and Neu5Gc. Amino-acid sequence and structural comparison of the active site of VcCMAS with those of eukaryotic and other bacterial counterparts reveal a diverse hydrophobic pocket that interacts with the C5 substituents of sialic acid. Analyses of the thermodynamic signatures obtained from the binding of the nucleotide (CTP) and the product (CMP-sialic acid) to VcCMAS provide fundamental information on the energetics of the binding process., (open access.)

Sortases are cysteine transpeptidases that assemble surface proteins and pili in their cell envelope. Encoded by all Gram-positive bacteria, few Gram-negative bacteria and archaea, sortases are currently divided into six classes (A-F). Due to the steep increase in bacterial genome data in recent years, the number of sortase homologues have also escalated rapidly. In this study, we used protein sequence similarity networks to explore the taxonomic diversity of sortases and also to evaluate the current classification of these enzymes. The resultant data suggest that sortase classes A, B, and D predominate in Firmicutes and classes E and F are enriched in Actinobacteria, whereas class C is distributed in both Firmicutes and Actinobacteria except Streptomyces family. Sortases were also observed in various Gram-negatives and euryarchaeota, which should be recognized as novel classes of sortases. Motif analysis around the catalytic cysteine was also performed and suggested that the residue at 2 nd position from cysteine may help distinguish various sortase classes. Moreover, the sequence analysis indicated that the catalytic arginine is highly conserved in almost all classes except sortase F in which arginine is replaced by asparagine in Actinobacteria. Additionally, class A sortases showed higher structural variation as compared to other sortases, whereas inter-class comparisons suggested structures of class C and D2 exhibited best similarities. A better understanding of the residues highlighted in this study should be helpful in elucidating their roles in substrate binding and the sortase function, and successively could help in the development of strong sortase inhibitors.

Tannerella forsythia is a gram-negative anaerobic bacterium that is associated with the development of destructive periodontal disease. T. forsythia secretes the metalloprotease-like enzyme karilysin. Using in vitro systems karilysin has been shown to modulate the host immune response by degradation of complement system proteins and by inactivation of the antimicrobial peptide LL-37 by proteolytic cleavage. This makes karilysin a highly interesting virulence factor to study in the framework of drug development and diagnostics. However, to date the presence of karilysin in clinical samples has not been demonstrated due to the lack of specific probes. In the present work, a high titer and stable affinity-purified avian IgY antibody against karilysin was developed. By surface plasmon resonance imaging the IgY affinity was found to be in the low nanomolar range. The antibody could be used to detect karilysin in saliva samples by immuno-blotting and was specific when tested towards human MMP-3. Furthermore, an avian IgY-based immunoassay was developed, which demonstrated low intra- and interday assay variability (CV's below 10%). Application of the immunoassay on a well-characterized set of saliva samples from adolescents with or without signs of periodontitis showed that it was possible to detect karilysin in saliva. A significant difference in karilysin concentration was found between saliva from participants with signs of periodontitis and saliva from healthy controls (p = .0024). The median of karilysin levels among periodontitis cases was 957 pg/ml (IQR, 499-2132 pg/ml) and the median for controls was 569 pg/ml (IQR, 210-1343 pg/ml). Collectively our data confirm the presence of karilysin in clinical samples. The described IgY-based immunoassay may prove useful as part of protein-based biomarker screenings in the clinic or in point-of care settings., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Mycobacterium tuberculosis (MTB) is a common bacterium causing tuberculosis and remains a major pathogen for mortality. Although the MTB genome has been extensively explored for two decades, the functions of 27% (1051/3906) of encoded proteins have yet to be determined and these proteins are annotated as hypothetical proteins., Methods: We assigned functions to these hypothetical proteins using SSEalign, a newly designed algorithm utilizing structural information. A set of rigorous criteria was applied to these annotations in order to examine whether they were supported by each parameter. Virulence factors and potential drug targets were also screened among the annotated proteins., Results: For 78% (823/1051) of the hypothetical proteins, we could identify homologs in Escherichia coli and Salmonella typhimurium by using SSEalign. Functional classification analysis indicated that 62.2% (512/823) of these annotated proteins were enzymes with catalytic activities and most of these annotations were supported by at least two other independent parameters. A relatively high proportion of transporter was identified in MTB genome, indicating the potential frequent transportation of frequent absorbing essential metabolites and excreting toxic materials in MTB. Twelve virulence factors and ten vaccine candidates were identified within these MTB hypothetical proteins, including two genes (rpoS and pspA) related to stress response to the host immune system. Furthermore, we have identified six novel drug target candidates among our annotated proteins, including Rv0817 and Rv2927c, which could be used for treating MTB infection., Conclusions: Our annotation of the MTB hypothetical proteins will probably serve as a useful dataset for future MTB studies.