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<jats:title>Abstract</jats:title><jats:p>Acute respiratory infection by influenza virus is a persistent and pervasive public health problem. Antiviral innate immunity initiated by type I interferon (IFN) is the first responder to pathogen invasion and provides the first line of defense. We discovered that Axin1, a scaffold protein, was reduced during influenza virus infection. We also found that overexpression of Axin1 and the chemical stabilizer of Axin1, XAV939, reduced influenza virus replication in lung epithelial cells. This effect was also observed with respiratory syncytial virus and vesicular stomatitis virus. Axin1 boosted type I IFN response to influenza virus infection and activated JNK/c‐Jun and Smad3 signaling. XAV939 protected mice from influenza virus infection. Thus, our studies provide new mechanistic insights into the regulation of the type I IFN response and present a new potential therapeutic of targeting Axin1 against influenza virus infection.</jats:p>

<jats:title>Abstract</jats:title><jats:p><jats:italic>Coxiella burnetii</jats:italic> is the causative agent of Q fever. All <jats:italic>C. burnetii</jats:italic> isolates encode either an autonomously replicating plasmid (QpH1, QpDG, QpRS, or QpDV) or QpRS‐like chromosomally integrated plasmid sequences. The role of the ORFs present in these sequences is unknown. Here, the role of the ORFs encoded on QpH1 was investigated. Using a new <jats:italic>C. burnetii</jats:italic> shuttle vector (pB‐TyrB‐QpH1ori), we cured the <jats:italic>C. burnetii</jats:italic> Nine Mile Phase II strain of QpH1. The ΔQpH1 strain grew normally in axenic media but had a significant growth defect in Vero cells, indicating QpH1 was important for <jats:italic>C. burnetii</jats:italic> virulence. We developed an inducible CRISPR interference system to examine the role of individual QpH1 plasmid genes. CRISPRi of <jats:italic>cbuA0027</jats:italic> resulted in significant growth defects in axenic media and THP‐1 cells. The <jats:italic>cbuA0028</jats:italic>/<jats:italic>cbuA0027</jats:italic> operon encodes CBUA0028 (ToxP) and CBUA0027 (AntitoxP), which are homologous to the HigB2 toxin and HigA2 antitoxin, respectively, from <jats:italic>Vibrio cholerae</jats:italic>. Consistent with toxin–antitoxin systems, overexpression of <jats:italic>toxP</jats:italic> resulted in a severe intracellular growth defect that was rescued by co‐expression of <jats:italic>antitoxP</jats:italic>. ToxP inhibited protein translation. AntitoxP bound the <jats:italic>toxP</jats:italic> promoter (P<jats:italic>toxP</jats:italic>) and ToxP, with the resulting complex binding also P<jats:italic>toxP</jats:italic>. In summary, our data indicate that <jats:italic>C. burnetii</jats:italic> maintains an autonomously replicating plasmid because of a plasmid‐based toxin–antitoxin system.</jats:p>

<jats:title>Abstract</jats:title><jats:p><jats:italic>Enterococcus faecalis</jats:italic> virulence requires cell wall‐associated proteins, including the sortase‐assembled endocarditis and biofilm associated pilus (Ebp), important for biofilm formation in vitro and in vivo. The current paradigm for sortase‐assembled pilus biogenesis in Gram‐positive bacteria is that sortases attach substrates to lipid II peptidoglycan (PG) precursors, prior to their incorporation into the growing cell wall. Contrary to prevailing dogma, by following the distribution of Ebp and PG throughout the <jats:italic>E. faecalis</jats:italic> cell cycle, we found that cell surface Ebp do not co‐localize with newly synthesized PG. Instead, surface‐exposed Ebp are localized to the older cell hemisphere and excluded from sites of new PG synthesis at the septum. Moreover, Ebp deposition on the younger hemisphere of the <jats:italic>E. faecalis</jats:italic> diplococcus appear as foci adjacent to the nascent septum. We propose a new model whereby sortase substrate deposition can occur on older PG rather than at sites of new cell wall synthesis. Consistent with this model, we demonstrate that sequestering lipid II to block PG synthesis via ramoplanin, does not impact new Ebp deposition at the cell surface. These data support an alternative paradigm for sortase substrate deposition in <jats:italic>E. faecalis</jats:italic>, in which Ebp are anchored directly onto uncrosslinked cell wall, independent of new PG synthesis.</jats:p>

<jats:title>Abstract</jats:title><jats:p>The outer membrane (OM) of Gram‐negative bacteria functions as an essential barrier and is characterized by an asymmetric bilayer with lipopolysaccharide (LPS) in the outer leaflet. The enzyme LpxC catalyzes the first committed step in LPS biosynthesis. It plays a critical role in maintaining the balance between LPS and phospholipids (PL), which are both derived from the same biosynthetic precursor. The essential inner membrane proteins YejM (PbgA, LapC), LapB (YciM), and the protease FtsH are known to account for optimal LpxC levels, but the mechanistic details are poorly understood. LapB is thought to be a bi‐functional protein serving as an adaptor for FtsH‐mediated turnover of LpxC and acting as a scaffold in the coordination of LPS biosynthesis. Here, we provide experimental evidence for the physical interaction of LapB with proteins at the biosynthetic node from where the LPS and PL biosynthesis pathways diverge. By a total of four in vivo and in vitro assays, we demonstrate protein–protein interactions between LapB and the LPS biosynthesis enzymes LpxA, LpxC, and LpxD, between LapB and YejM, the anti‐adaptor protein regulating LapB activity, and between LapB and FabZ, the first PL biosynthesis enzyme. Moreover, we uncovered a new adaptor function of LapB in destabilizing not only LpxC but also LpxD. Overall, our study shows that LapB is a multi‐functional protein that serves as a protein–protein interaction hub for key enzymes in LPS and PL biogenesis presumably by virtue of multiple tetratricopeptide repeat (TPR) motifs in its cytoplasmic C‐terminal region.</jats:p>

<jats:title>Abstract</jats:title><jats:p><jats:italic>Escherichia coli</jats:italic> has multiple pathways to release nonproductive ribosome complexes stalled at the 3′ end of nonstop mRNA: tmRNA (SsrA RNA)‐mediated <jats:italic>trans</jats:italic>‐translation and stop codon‐independent termination by ArfA/RF2 or ArfB (YaeJ). The <jats:italic>arfA</jats:italic> mRNA lacks a stop codon and its expression is repressed by <jats:italic>trans</jats:italic>‐translation. Therefore, ArfA is considered to complement the ribosome rescue activity of <jats:italic>trans</jats:italic>‐translation, but the physiological situations in which ArfA is expressed have not been elucidated. Here, we found that the excision of CP4‐57 prophage adjacent to <jats:italic>E. coli ssrA</jats:italic> leads to the inactivation of tmRNA and switches the primary rescue pathway from <jats:italic>trans</jats:italic>‐translation to ArfA/RF2. This “rescue‐switching” rearranges not only the proteome landscape in <jats:italic>E. coli</jats:italic> but also the phenotype such as motility. Furthermore, among the proteins with significantly increased abundance in the ArfA<jats:sup>+</jats:sup> cells, we found ZntR, whose mRNA is transcribed together as the upstream part of nonstop <jats:italic>arfA</jats:italic> mRNA. Repression of ZntR and reconstituted model genes depends on the translation of the downstream nonstop ORFs that trigger the <jats:italic>trans</jats:italic>‐translation‐coupled exonucleolytic degradation by polynucleotide phosphorylase (PNPase). Namely, our studies provide a novel example of <jats:italic>trans</jats:italic>‐translation‐dependent regulation and re‐define the physiological roles of prophage excision.</jats:p>

<jats:title>Abstract</jats:title><jats:p>The marine pathogen <jats:italic>Vibrio vulnificus</jats:italic> senses and responds to environmental stimuli via two chemosensory systems and 42–53 chemoreceptors. Here, we present an analysis of the <jats:italic>V. vulnificus</jats:italic> Aer2 chemoreceptor, <jats:italic>Vv</jats:italic>Aer2, which is the first <jats:italic>V. vulnificus</jats:italic> chemoreceptor to be characterized. <jats:italic>Vv</jats:italic>Aer2 is related to the Aer2 receptors of other gammaproteobacteria, but uncharacteristically contains three PAS domains (PAS1‐3), rather than one or two. Using an <jats:italic>E. coli</jats:italic> chemotaxis hijack assay, we determined that <jats:italic>Vv</jats:italic>Aer2, like other Aer2 receptors, senses and responds to O<jats:sub>2</jats:sub>. All three <jats:italic>Vv</jats:italic>Aer2 PAS domains bound pentacoordinate <jats:italic>b‐</jats:italic>type heme and exhibited similar O<jats:sub>2</jats:sub> affinities. PAS2 and PAS3 both stabilized O<jats:sub>2</jats:sub> via conserved Iβ‐Trp residues, but PAS1, which was easily oxidized in vitro, was unaffected by Iβ‐Trp replacement. Our results support a model in which PAS1 is largely dispensable for O<jats:sub>2</jats:sub>‐mediated signaling, whereas PAS2 modulates PAS3 signaling, and PAS3 signals to the downstream domains. Each PAS domain appeared to be positionally optimized, because PAS swapping caused altered signaling properties, and neither PAS1 nor PAS2 could replace PAS3. Our findings strengthen previous conclusions that Aer2 receptors are O<jats:sub>2</jats:sub> sensors, but with distinct N‐terminal domain arrangements that facilitate, modulate and tune responses based on environmental signals.</jats:p>

<jats:title>Abstract</jats:title><jats:p>Mammalian professional phagocytic cells ingest and kill invading microorganisms and prevent the development of bacterial infections. Our understanding of the sequence of events that results in bacterial killing and permeabilization in phagosomes is still largely incomplete. In this study, we used the <jats:italic>Dictyostelium discoideum</jats:italic> amoeba as a model phagocyte to study the fate of the bacteria <jats:italic>Klebsiella pneumoniae</jats:italic> inside phagosomes. Our analysis distinguishes three consecutive phases: bacteria first lose their ability to divide (killing), then their cytosolic content is altered (permeabilization), and finally their DNA is degraded (digestion). Phagosomal acidification and production of free radicals are necessary for rapid killing, membrane‐permeabilizing proteins BpiC and AlyL are required for efficient permeabilization. These results illustrate how a combination of genetic and microscopical tools can be used to finely dissect the molecular events leading to bacterial killing and permeabilization in a maturing phagosome.</jats:p>

<jats:title>Abstract</jats:title><jats:p>Methionine is a sulfur‐containing residue found in most proteins which are particularly susceptible to oxidation. Although methionine oxidation causes protein damage, it can in some cases activate protein function. Enzymatic systems reducing oxidized methionine have evolved in most bacterial species and methionine oxidation proves to be a reversible post‐translational modification regulating protein activity. In this review, we inspect recent examples of methionine oxidation provoking protein loss and gain of function. We further speculate on the role of methionine oxidation as a multilayer endogenous antioxidant system and consider its potential consequences for bacterial virulence.</jats:p>

<jats:title>Abstract</jats:title><jats:p><jats:italic>Actinobacteria</jats:italic> have a complex life cycle, including morphological and physiological differentiation which are often associated with the biosynthesis of secondary metabolites. Recently, increased interest in post‐translational modifications (PTMs) in these Gram‐positive bacteria has highlighted the importance of PTMs as signals that provide functional diversity and regulation by modifying proteins to respond to diverse stimuli. Here, we review the developments in research on acylation, a typical PTM that uses acyl‐CoA or related metabolites as donors, as well as the understanding of the direct link provided by acylation between cell metabolism and signal transduction, transcriptional regulation, cell growth, and pathogenicity in <jats:italic>Actinobacteria</jats:italic>.</jats:p>

<jats:title>Abstract</jats:title><jats:p><jats:italic>Streptococcus pneumoniae</jats:italic> has to cope with the strong oxidant hypochlorous acid (HOCl), during host–pathogen interactions. Thus, we analyzed the global gene expression profile of <jats:italic>S. pneumoniae</jats:italic> D39 towards HOCl stress. In the RNA‐seq transcriptome, the NmlR, SifR, CtsR, HrcA, SczA and CopY regulons and the <jats:italic>etrx1‐ccdA1‐msrAB2</jats:italic> operon were most strongly induced under HOCl stress, which participate in the oxidative, electrophile and metal stress response in <jats:italic>S. pneumoniae</jats:italic>. The MerR‐family regulator NmlR harbors a conserved Cys52 and controls the alcohol dehydrogenase‐encoding <jats:italic>adhC</jats:italic> gene under carbonyl and NO stress. We demonstrated that NmlR senses also HOCl stress to activate transcription of the <jats:italic>nmlR‐adhC</jats:italic> operon. HOCl‐induced transcription of <jats:italic>adhC</jats:italic> required Cys52 of NmlR in vivo. Using mass spectrometry, NmlR was shown to be oxidized to intersubunit disulfides or <jats:italic>S</jats:italic>‐glutathionylated under oxidative stress in vitro. A broccoli‐FLAP‐based assay further showed that both NmlR disulfides significantly increased transcription initiation at the <jats:italic>nmlR</jats:italic> promoter by RNAP in vitro, which depends on Cys52. Phenotype analyses revealed that NmlR functions in the defense against oxidative stress and promotes survival of <jats:italic>S. pneumoniae</jats:italic> during macrophage infections<jats:italic>.</jats:italic> In conclusion, NmlR was characterized as HOCl‐sensing transcriptional regulator, which activates transcription of <jats:italic>adhC</jats:italic> under oxidative stress by thiol switches in <jats:italic>S. pneumoniae</jats:italic>.</jats:p>