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Salmonella are a type of bacteria known to cause food-borne illness. Their host range varies widely, and their susceptibility to the host determines its pathogenicity. Salmonella enterica serovar Rissen (S. Rissen) is a widely distributed serotype; however, its virulence and pathogenicity are poorly understood. In this study, the pathogenicity and antibiotic resistance of a representative S. Rissen isolate were investigated. The cell model results showed that S. Rissen preferred to replicate in human macrophage cells U937 compared to murine macrophage cells RAW264.7, suggesting that it has a level of host adaptability. Genome sequencing and comparison analysis revealed that the distribution and nonsynonymous single nucleotide polymorphisms of virulence factors in S. Rissen were similar to those in S. Typhi rather than to those in S. Typhimurium. Taken together, our results suggest that although S. Rissen is a common serotype distributed in swine herds, pork and chicken products, it has strong ability to infect humans.

The two-component system PhoP/PhoQ is essential for Salmonella enterica serovar Typhimurium virulence. Here, we report that PhoP is methylated extensively. Two consecutive glutamate (E) and aspartate (D)/E residues, i.e., E8/D9 and E107/E108, and arginine (R) 112 can be methylated. Individual mutation of these above-mentioned residues caused impaired phosphorylation and dimerization or DNA-binding ability of PhoP to a different extent and led to attenuated bacterial virulence. With the help of specific antibodies recognizing methylated E8 and monomethylated R112, we found that the methylation levels of E8 or R112 decreased dramatically when bacteria encountered low magnesium, acidic pH, or phagocytosis by macrophages, under which PhoP can be activated. Furthermore, CheR, a bacterial chemotaxis methyltransferase, was identified to methylate R112. Overexpression of cheR decreased PhoP activity but increased PhoP stability. Together, the current study reveals that methylation plays an important role in regulating PhoP activities in response to environmental cues and, consequently, modulates Salmonella virulence. IMPORTANCE Posttranslational modifications (PTMs) play an important role in regulating enzyme activities, protein-protein interactions, or DNA-protein recognition and, consequently, modulate many biological functions. We demonstrated that PhoP, the response regulator of PhoP/PhoQ two-component system, could be methylated on several evolutionally conserved amino acid residues. These amino acid residues were crucial for PhoP phosphorylation or dimerization, DNA-binding ability of PhoP, and Salmonella virulence. Interestingly, methylation negatively regulated the activity of PhoP. A bacterial chemotaxis methyltransferase CheR was involved in PhoP methylation. Methylation of PhoP could stabilize it in an inactive conformation. Our work provides a more informative depiction of PhoP PTM and markedly improves our understanding of the coordinate regulation of bacterial chemotaxis and virulence.

The PhoP-PhoQ two-component regulation system of Salmonella enterica serovar Typhimurium is involved in the response to various environmental stresses and is essential for bacterial virulence. Our previous studies showed that acetylation plays an important role in regulating the activity of PhoP, which consequently mediates the change in virulence of S Typhimurium. Here, we demonstrate that a conserved lysine residue, K88, is crucial for the function of PhoP and its acetylation-downregulated PhoP activities. K88 could be specifically acetylated by acetyl phosphate (AcP), and the acetylation level of K88 decreased significantly after phagocytosis of S Typhimurium by macrophages. Acetylation of K88 inhibited PhoP dimerization and DNA-binding abilities. In addition, mutation of K88 to glutamine, mimicking the acetylated form, dramatically attenuated intestinal inflammation and systemic infection of S Typhimurium in the mouse model. These findings indicate that nonenzymatic acetylation of PhoP by AcP is a fine-tuned mechanism for the virulence of S Typhimurium and highlights that virulence and metabolism in the host are closely linked.

The PhoP-PhoQ two-component regulation system of Salmonella enterica serovar Typhimurium is involved in the response to various environmental stresses and is essential for bacterial virulence. Our previous studies showed that acetylation plays an important role in regulating the activity of PhoP, which consequently mediates the change in virulence of S. Typhimurium. Here, we demonstrate that a conserved lysine residue, K88, is crucial for the function of PhoP and its acetylation-downregulated PhoP activities. K88 could be specifically acetylated by acetyl phosphate (AcP), and the acetylation level of K88 decreased significantly after phagocytosis of S. Typhimurium by macrophages. Acetylation of K88 inhibited PhoP dimerization and DNA-binding abilities. In addition, mutation of K88 to glutamine, mimicking the acetylated form, dramatically attenuated intestinal inflammation and systemic infection of S. Typhimurium in the mouse model. These findings indicate that nonenzymatic acetylation of PhoP by AcP is a fine-tuned mechanism for the virulence of S. Typhimurium and highlights that virulence and metabolism in the host are closely linked.

Mycobacterium tuberculosis (M.tb) is one of the most prevalent bacterial pathogens in the world. With geographical wide spread and hypervirulence, Beijing/W family is the most successful M.tb lineage. China is a country of high tuberculosis (TB) and high multiple drug-resistant TB (MDR-TB) burden, and the Beijing/W family strains take the largest share of MDR strains. To study the genetic basis of Beijing/W family strains' virulence and drug resistance, we performed the whole genome sequencing of M.tb strain W146, a clinical Beijing/W genotype MDR isolated from Wuxi, Jiangsu province, China. Compared with genome sequence of M.tb strain H37Rv, we found that strain W146 lacks three large fragments and the missing of furA-katG operon confers isoniazid resistance. Besides the missing of furA-katG operon, strain W146 harbored almost all known drug resistance-associated mutations. Comparison analysis of single nucleotide polymorphisms (SNPs) and indels between strain W146 and Beijing/W genotype strains and non-Beijing/W genotype strains revealed that strain W146 possessed some unique mutations, which may be related to drug resistance, transmission and pathogenicity. These findings will help to understand the large sequence polymorphisms (LSPs) and the transmission and drug resistance related genetic characteristics of the Beijing/W genotype of M.tb.

SIRT family proteins are highly conserved both in the structure and function among all the organisms, and are involved in gene silencing, DNA damage repair, cell growth and metabolism. Here, a SIRT4 homologue MSMEG_4620 was identified and characterized in Mycobacterium smegmatis. MSMEG_4620 exhibits deacetylase activity that can be activated by fatty acids. Interestingly, MSMEG_4620 also possesses auto ADP-ribosylation activity. MSMEG_4620 is modified on arginine residues as revealed by a chemical stability assay. Moreover, the auto ADP-ribosylation activity of MSMEG_4620 was found to be enhanced by ferric ion. Notably, the SIRT4 homologues are widely distributed in the genomes of environmental mycobacterial species instead of pathogenic mycobacterial species. When MSMEG_4620 was deleted in M. smegmatis, the mutant strain showed a growth defect in 7H9 minimal medium compared with the parental strain. Taken together, these results provided the characteristics of a SIRT4 homologue in prokaryotes and implicated its critical roles in the growth of environmental mycobacterial species.

The ability to acetylate lysine residues is conserved across organisms, and acetylation of lysine residues plays important roles in various cellular functions. Maintaining intracellular pH homeostasis is crucial for the survival of enteric bacteria in the acidic gastric tract. It has been shown that eukaryotes can stabilize the intracellular pH by histone deacetylation. However, it remains unknown whether bacteria can utilize a reversible protein acetylation system to adapt to an acidic environment. Here we demonstrate that protein acetylation/deacetylation is critical for Salmonella enterica serovar Typhimurium to survive in an acidic environment. We used RNA sequencing to analyze the transcriptome patterns under acid stress and found that the transcriptional levels of genes involved in NAD + /NADH metabolism were significantly changed, leading to an increase in the intracellular NAD + /NADH ratio. Moreover, acid stress downregulated the transcriptional level of pat , encoding acetyltransferase, and genes cyaA and crp , encoding adenylate cyclase and cyclic AMP receptor protein, respectively, which are positive regulators of pat . It was found that the acid signal alerts the tricarboxylic acid cycle to promote the consumption of acetyl coenzyme A (Ac-CoA), an acetyl group donor for the acetylation reaction. A lowered acetylation level not only was the bacterial response to acid stress but also increased the survival rate of S . Typhimurium under acid stress. The pat deletion mutant had a more stable intracellular pH, which paralleled the higher survival rate after acid treatment compared with that of both the wild-type strain and the cobB (encoding deacetylase) deletion mutant. Our data indicate that bacteria can downregulate the protein acetylation level to prevent the intracellular pH from further falling under acid stress, and this work may provide a new perspective to understand the bacterial acid resistance mechanism.

The aim of this work was to screen miRNA signatures dysregulated in tuberculosis to improve our understanding of the biological role of miRNAs involved in the disease. Datasets deposited in publically available databases from microarray studies on infectious diseases and malignancies were retrieved, screened, and subjected to further analysis. Effect sizes were combined using the inverse-variance model and between-study heterogeneity was evaluated by the random effects model. 35 miRNAs were differentially expressed (12 up-regulated, 23 down-regulated; p

Tuberculosis (TB) is a communicable disease of major global importance and causes metabolic disorder of the patients. In a previous study, we found that the plasma metabolite profile of TB patients differs from that of healthy control subjects based on nuclear magnetic resonance (NMR) spectroscopy. In order to evaluate the TB specificity of the metabolite profile, a total of 110 patients, including 40 with diabetes, 40 with malignancy, and 30 with community-acquired pneumonia (CAP), assessed by NMR spectroscopy, and compared to those of patients with TB. Based on the orthogonal partial least-squares discriminant analysis (OPLS-DA), the metabolic profiles of these diseases were significant different, as compared to the healthy controls and TB patients, respectively. The score plots of the OPLS-DA model demonstrated that TB was easily distinguishable from diabetes, CAP and malignancy. Plasma levels of ketone bodies, lactate, and pyruvate were increased in TB patient compared to healthy control, but lower than CAP and malignancy. We conclude that the metabolic profiles were TB-specific and reflected MTB infection. Our results strongly support the NMR spectroscopy-based metabolomics could contribute to an improved understanding of disease mechanisms and may offer clues to new TB clinic diagnosis and therapies.

Nuclear magnetic resonance (NMR) spectroscopy is an ideal platform for the metabolic analysis of biofluids, due to its high reproducibility, non-destructiveness, non-selectivity in metabolite detection, and the ability to simultaneously quantify multiple classes of metabolites. Tuberculosis (TB) is a chronic wasting inflammatory disease characterized by multi-system involvement, which can cause metabolic derangements in afflicted patients. In this study, we combined multivariate pattern recognition (PR) analytical techniques with 1H NMR spectroscopy to explore the metabolic profile of sera from TB patients. A total of seventy-seven serum samples obtained from patients with TB (n=38) and healthy controls (n=39) were investigated. Orthogonal partial least-squares discriminant analysis (OPLS-DA) was capable of distinguishing TB patients from controls, and establishing a TB-specific metabolite profile. A total of 17 metabolites differed significantly in concentration between the two groups. Serum samples from TB patients were characterized by increased concentrations of 1-methylhistidine, acetoacetate, acetone, glutamate, glutamine, isoleucine, lactate, lysine, nicotinate, phenylalanine, pyruvate, and tyrosine, accompanied by reduced concentrations of alanine, formate, glycine, glycerolphosphocholine, and low-density lipoproteins relative to control subjects. Our study reveals the metabolic profile of sera from TB patients and indicates that NMR-based methods can distinguish TB patients from healthy controls. NMR-based metabolomics has the potential to be developed into a novel clinical tool for TB diagnosis and/or therapeutic monitoring, and could contribute to an improved understanding of disease mechanisms.

Enterohemorrhagic Escherichia coli (EHEC) is one major type of contagious and foodborne pathogens. The type VI secretion system (T6SS) has been shown to be involved in the bacterial pathogenicity and bacteria-bacteria competition. Here, we show that EHEC could secrete a novel effector KatN, a Mn-containing catalase, in a T6SS-dependent manner. Expression of katN is promoted by RpoS and OxyR and repressed by H-NS, and katN contributes to bacterial growth under oxidative stress in vitro. KatN could be secreted into host cell cytosol after EHEC is phagocytized by macrophage, which leads to decreased level of intracellular reactive oxygen species (ROS) and facilitates the intramacrophage survival of EHEC. Finally, animal model results show that the deletion mutant of T6SS was attenuated in virulence compared with the wild type strain, while the deletion mutant of katN had comparable virulence to the wild type strain. Taken together, our findings suggest that EHEC could sense oxidative stress in phagosome and decrease the host cell ROS by secreting catalase KatN to facilitate its survival in the host cells., Author summary The type VI secretion system (T6SS) is a specific macromolecular protein export apparatus, and widely distributed in Gram-negative bacteria. Generally, T6SS has been shown to play an important role in anti-bacterial competition and virulence to eukaryotic hosts. Enterohemorrhagic Escherichia coli (EHEC) can cause severe foodborne disease, including abdominal cramps and diarrhea that may progress to bloody diarrhea and hemolytic uremic syndrome. In the current study, we show that the T6SS of EHEC is involved in its intracellular survival and virulence in mice. Specifically, the novel effector KatN, a Mn-catalase identified in this work updates the general role of the T6SS in the pathogenesis of EHEC, in the context of oxidative stress in host cytoplasm. Combined with the biased distribution of katN in the T6SS-containing bacteria, our data suggest that KatN, as a new T6SS effector, is a key virulence factor in the pathogenesis of T6SS+ bacteria.

The regulation of chromosomal replication is critical and the activation of DnaA by ATP binding is a key step in replication initiation. However, it remains unclear whether and how the process of ATP-binding to DnaA is regulated. Here, we show that DnaA can be acetylated and its acetylation level varies with cell growth and correlates with DNA replication initiation frequencies in E. coli. Specifically, the conserved K178 in Walker A motif of DnaA can be acetylated and its acetylation level reaches the summit at the stationary phase, which prevents DnaA from binding to ATP or oriC and leads to inhibition of DNA replication initiation. The deacetylation process of DnaA is catalyzed by deacetylase CobB. The acetylation process of DnaA is mediated by acetyltransferase YfiQ and nonenzymatically by acetyl-phosphate. These findings suggest that the reversible acetylation of DnaA ensures cells to respond promptly to environmental changes. Since Walker A motif is universally distributed across organisms, acetylation of Walker A motif may present a novel regulatory mechanism conserved from bacteria to eukaryotes.

As a global transcriptional regulator, H-NS, the histone-like nucleoid-associated DNA-binding and bridging protein, plays a wide range of biological roles in bacteria. In order to determine the role of H-NS in regulating gene transcription and further find out the biological significance of this protein in Enterohemorrhagic Escherichia coli (EHEC), we conducted transcriptome analysis of hns mutant by RNA sequencing. A total of 983 genes were identified to be regulated by H-NS in EHEC. 213 and 770 genes were down-regulated and up-regulated in the deletion mutant of hns, respectively. Interestingly, 34 of 97 genes on virulence plasmid pO157 were down-regulated by H-NS. Although the deletion mutant of hns showed a decreased survival rate in macrophage compared with the wild type strain, it exhibited the higher ability to colonize mice gut and became more virulent to BALB/c mice. The BALB/c mice infected with the deletion mutant of hns showed a lower survival rate, and a higher bacterial burden in the gut, compared with those infected with wild type strain, especially when the gut microbiota was not disturbed by antibiotic administration. These findings suggest that H-NS plays an important role in virulence of EHEC by interacting with host gut microbiota.

Cellular inhibitor of apoptosis protein 2 (cIAP2) is a potent suppressor of apoptotic cell death. We have shown previously that cIAP2 is involved in the tumor necrosis factor alpha (TNF-α)-induced anti-hepatitis B virus (HBV) response; however, the mechanism for this antiviral effect remains unclear. In the present study, we demonstrate that cIAP2 can significantly reduce the levels of HBV DNA replication intermediates but not the total viral RNA or core protein levels. Domain-mapping analysis revealed that the carboxy-terminal domains of cIAP2 were indispensable for this anti-HBV ability and that an E3 ligase-deficient mutant of cIAP2 (termed cIAP2*) completely lost its antiviral activity. We further identified HBV polymerase as the target of cIAP2. Overexpression of cIAP2 but not cIAP2* reduced polymerase protein levels, while cIAP2 knockdown increased polymerase expression. In addition, we observed that cIAP2 promoted the degradation of the viral polymerase through a proteasome-dependent pathway. Further experiments demonstrated that cIAP2 can bind to polymerase and promote its polyubiquitylation. Finally, we found that cIAP2 downregulated the encapsidation of HBV pregenomic RNA. Taken together, these data reveal a novel mechanism for the inhibition of HBV replication by cIAP2 via acceleration of the ubiquitin-proteasome-mediated decay of polymerase and reduction of the encapsidation of HBV pregenomic RNA, making this mechanism a novel strategy for HBV therapy.

SIRT family proteins are highly conserved both in the structure and function among all the organisms, and are involved in gene silencing, DNA damage repair, cell growth and metabolism. Here, a SIRT4 homologue MSMEG_4620 was identified and characterized in Mycobacterium smegmatis. MSMEG_4620 exhibits deacetylase activity that can be activated by fatty acids. Interestingly, MSMEG_4620 also possesses auto ADP-ribosylation activity. MSMEG_4620 is modified on arginine residues as revealed by a chemical stability assay. Moreover, the auto ADP-ribosylation activity of MSMEG_4620 was found to be enhanced by ferric ion. Notably, the SIRT4 homologues are widely distributed in the genomes of environmental mycobacterial species instead of pathogenic mycobacterial species. When MSMEG_4620 was deleted in M. smegmatis, the mutant strain showed a growth defect in 7H9 minimal medium compared with the parental strain. Taken together, these results provided the characteristics of a SIRT4 homologue in prokaryotes and implicated its critical roles in the growth of environmental mycobacterial species.

Salmonella causes a range of diseases in different hosts, including enterocolitis and systemic infection. Lysine acetylation regulates many eukaryotic cellular processes, but its function in bacteria is largely unexplored. The acetyltransferase Pat and NAD(+)-dependent deacetylase CobB are involved in the reversible protein acetylation in Salmonella Typhimurium. Here, we used cell and animal models to evaluate the virulence of pat and cobB deletion mutants in S. Typhimurium and found that pat is critical for bacterial intestinal colonization and systemic infection. Next, to understand the underlying mechanism, genome-wide transcriptome was analyzed. RNA sequencing data showed that the expression of Salmonella pathogenicity island 1 (SPI-1) is partially dependent on pat In addition, we found that HilD, a key transcriptional regulator of SPI-1, is a substrate of Pat. The acetylation of HilD by Pat maintained HilD stability and was essential for the transcriptional activation of HilA. Taken together, these results suggest that a protein acetylation system regulates SPI-1 expression by controlling HilD in a posttranslational manner to mediate S. Typhimurium virulence.

Lipoprotein NlpI of Escherichia coli is involved in the cell division, virulence, and bacterial interaction with eukaryotic host cells. To elucidate the functional mechanism of NlpI, we examined how NlpI affects cell division and found that induction of NlpI inhibits nucleoid division and halts cell growth. Consistent with these results, the cell division protein FtsZ failed to localize at the septum but diffused in the cytosol. Elevation of NlpI expression enhanced the transcription and the outer membrane localization of the heat shock protein IbpA and IbpB. Deletion of either ibpA or ibpB abolished the effects of NlpI induction, which could be restored by complementation. The C-terminus of NlpI is critical for the enhancement in IbpA and IbpB production, and the N-terminus of NlpI is required for the outer membrane localization of NlpI, IbpA, and IbpB. Furthermore, NlpI physically interacts with IbpB. These results indicate that over-expression of NlpI can interrupt the nucleoids division and the assembly of FtsZ at the septum, mediated by IbpA/IbpB, suggesting a role of the NlpI/IbpA/IbpB complex in the cell division.

In this paper, we employ evolutionary game theory to analyze outsourcing vendors’ collaborative and competitive behavior. A game model among these contractors is given together with the payoff matrixes, and replicator dynamic equations and evolutionary stable strategies are discussed. We also introduce supervision and penalty mechanism into the game model and the analysis show that appropriate mechanism can make collaborative behaviors of concerned contractors an evolutionary stable strategy.

Type VI secretion systems (T6SSs) are involved in the pathogenicity of several Gram-negative bacteria. Based on sequence analysis, we found that a cluster of E scherichia coli v irulence f actors (EVF) encoding a putative T6SS exists in the genome of the meningitis-causing E. coli K1 strain RS218. The T6SS-associated deletion mutants exhibited significant defects in binding to and invasion of human brain microvascular endothelial cells (HBMEC) compared with the parent strain. Hcp family proteins (the hallmark of T6SS), including Hcp1 and Hcp2, were localized in the bacterial outer membrane, but the involvements of Hcp1 and Hcp2 have been shown to differ in E. coli -HBMEC interaction. The deletion mutant of hcp2 showed defects in the bacterial binding to and invasion of HBMEC, while Hcp1 was secreted in a T6SS-dependent manner and induced actin cytoskeleton rearrangement, apoptosis, and the release of interleukin-6 (IL-6) and IL-8 in HBMEC. These findings demonstrate that the T6SS is functional in E. coli K1, and two Hcp family proteins participate in different steps of E. coli interaction with HBMEC in a coordinate manner, e.g., binding to and invasion of HBMEC, the cytokine and chemokine release followed by cytoskeleton rearrangement, and apoptosis in HBMEC. This is the first demonstration of the role of T6SS in meningitis-causing E. coli K1, and T6SS-associated Hcp family proteins are likely to contribute to the pathogenesis of E. coli meningitis.