Combined antimicrobial effect of two peptide nucleic acids against Staphylococcus aureus and S. pseudintermedius veterinary isolates.

BACKGROUND: Staphylococcus aureus and S. pseudintermedius are the major etiological agents of staphylococcal infections in humans, livestock, and companion animals. The misuse of antimicrobial drugs has led to the emergence of antimicrobial-resistant Staphylococcus spp., including methicillin-resistant S. aureus (MRSA) and methicillin-resistant S. pseudintermedius (MRSP). One novel therapeutic approach against MRSA and MRSP is a peptide nucleic acid (PNA) that can bind to the target nucleotide strands and block expression. Previously, two PNAs conjugated with cell-penetrating peptides (P-PNAs), antisense PNA (ASP)-cmk and ASP-deoD, targeting two essential genes in S. aureus, were constructed, and their antibacterial activities were analyzed. OBJECTIVES: This study analyzed the combined antibacterial effects of P-PNAs on S. aureus and S. pseudintermedius clinical isolates. METHODS: S. aureus ATCC 29740 cells were treated simultaneously with serially diluted ASP-cmk and ASP-deoD, and the minimal inhibitory concentrations (MICs) were measured. The combined P-PNA mixture was then treated with S. aureus and S. pseudintermedius veterinary isolates at the determined MIC, and the antibacterial effect was examined. RESULTS: The combined treatment of two P-PNAs showed higher antibacterial activity than the individual treatments. The MICs of two individual P-PNAs were 20 and 25 µM, whereas that of the combined treatment was 10 µM. The application of a combined treatment to clinical Staphylococcus spp. revealed S. aureus isolates to be resistant to P-PNAs and S. pseudintermedius isolates to be susceptible. CONCLUSIONS: These observations highlight the complexity of designing ASPs with high efficacy for potential applications in treating staphylococcal infections in humans and animals.

Development of antisense peptide-peptide nucleic acids against fluoroquinolone-resistant Escherichia coli.

BACKGROUND: Fluoroquinolones (FQs) are potent and broad-spectrum antibiotics commonly used to treat MDR bacterial infections, but bacterial resistance to FQs has emerged and spread rapidly around the world. The mechanisms for FQ resistance have been revealed, including one or more mutations in FQ target genes such as DNA gyrase (gyrA) and topoisomerase IV (parC). Because therapeutic treatments for FQ-resistant bacterial infections are limited, it is necessary to develop novel antibiotic alternatives to minimize or inhibit FQ-resistant bacteria. OBJECTIVES: To examine the bactericidal effect of antisense peptide-peptide nucleic acids (P-PNAs) that can block the expression of DNA gyrase or topoisomerase IV in FQ-resistant Escherichia coli (FRE). METHODS: A set of antisense P-PNA conjugates with a bacterial penetration peptide were designed to inhibit the expression of gyrA and parC and were evaluated for their antibacterial activities. RESULTS: Antisense P-PNAs, ASP-gyrA1 and ASP-parC1, targeting the translational initiation sites of their respective target genes significantly inhibited the growth of the FRE isolates. In addition, ASP-gyrA3 and ASP-parC2, which bind to the FRE-specific coding sequence within the gyrA and parC structural genes, respectively, showed selective bactericidal effects against FRE isolates. CONCLUSIONS: Our results demonstrate the potential of targeted antisense P-PNAs as antibiotic alternatives against FQ-resistance bacteria.

Exfoliated Single Layers of Layered Cobalt Hydroxide for Ultrafine Co3 O4 Nanoparticles on Graphene Nanosheets as an Efficient Electrocatalyst for Oxygen Reduction.

A highly effective way to produce an oxygen reduction electrocatalyst was developed through the self-assembly of exfoliated single layers of cobalt hydroxide (Co(OH)2 ) and graphene oxide (GO). These 2D materials have complete contact with one another because of their physical flexibility and the electrostatic attraction between negatively charged GO and positively charged Co(OH)2 layers. The strong coupling induces transformation of the Co(OH)2 single layer into a discrete nanocrystal of spinel Co3 O4 with an average size of 8 nm on reduced GO (RGO) during calcination, which could not be obtained with bulk-layered cobalt hydroxide because of its rapid layer collapse. The ultrafine Co3 O4 /RGO hybrid exhibited not only comparable performance in the oxygen reduction reaction but also higher durability compared with the commercial 20 wt % Pt/C catalyst.

A Novel Peptide Nucleic Acid against the Cytidine Monophosphate Kinase of S. aureus Inhibits Staphylococcal Infection In Vivo.

Here, we report a novel bactericidal peptide nucleic acid (PNA) that can induce the antisense effect on the cytidine monophosphate kinase (Cmk) of Staphylococcus aureus, a putative essential component for bacterial species. Based on the genome sequence of S. aureus N315, a set of PNA conjugates with a bacterial penetration peptide, (KFF)3K, were synthesized to target the seven potentially essential genes (cmk, deoD, ligA, smpB, glmU, pyrH, and ftsA) and further evaluated for their antibacterial properties in vitro as well as in vivo. The results demonstrated that two peptide-conjugated PNAs (P-PNAs), antisense P-PNA (ASP)-cmk1 and ASP-deoD1, targeting either the cmk or the deoD genes, had the strongest inhibitory effects on the growth of S. aureus ATCC 29740 (a bovine mastitic milk isolate) in a dose-dependent manner. In vivo application of ASP-cmk1 resulted in a significant reduction of bacterial loads in mice intraperitoneally infected with a sublethal dose of S. aureus. Moreover, ASP-cmk1 significantly increased the survival rate of the breast-fed infant mice after intramammary infection of the lactating CD-1 mice. Taken together, our characterization of ASP-cmk1 demonstrated its bactericidal activity against S. aureus as well as its effectiveness in vivo.

Antisense peptide nucleic acids as a potential anti-infective agent.

Antibiotics have long been used for anti-infective control of bacterial infections, growth promotion in husbandry, and prophylactic protection against plant pathogens. However, their inappropriate use results in the emergence and spread of multiple drug resistance (MDR) especially among various bacterial populations, which limits further administration of conventional antibiotics. Therefore, the demand for novel anti-infective approaches against MDR diseases becomes increasing in recent years. The peptide nucleic acid (PNA)-based technology has been proposed as one of novel anti-infective and/or therapeutic strategies. By definition, PNA is an artificially synthesized nucleic acid mimic structurally similar to DNA or RNA in nature and linked one another via an unnatural pseudo-peptide backbone, rendering to its stability in diverse host conditions. It can bind DNA or RNA strands complimentarily with high affinity and sequence specificity, which induces the target-specific gene silencing by inhibiting transcription and/or translation. Based on these unique properties, PNA has been widely applied for molecular diagnosis as well as considered as a potential anti-infective agent. In this review, we discuss the general features of PNAs and their application to various bacterial pathogens as new anti-infective or antimicrobial agents.

Pathogenic and phylogenetic characteristics of non-O157 Shiga toxin-producing Escherichia coli isolates from retail meats in South Korea.

Herein, we report the pathogenic and phylogenetic characteristics of seven Shiga toxin (Stx)-producing Escherichia coli (STEC) isolates from 434 retail meats collected in Korea during 2006 to 2012. The experimental analyses revealed that all isolates (i) were identified as non-O157 STEC, including O91:H14 (3 isolates), O121:H10 (2 isolates), O91:H21 (1 isolate), and O18:H20 (1 isolate), (ii) carried diverse Stx subtype genes (stx1, stx2c, stx2e, or stx1 + stx2b) whose expression levels varied strain by strain, and (iii) lacked the locus of enterocyte effacement (LEE) pathogenicity island, a major virulence factor of STEC, but they possessed one or more alternative virulence genes encoding cytotoxins (Cdt and SubAB) and/or adhesins (Saa, Iha, and EcpA). Notably, a significant heterogeneity in glutamate-induced acid resistance was observed among the STEC isolates (p ＜ 0.05). In addition, phylogenetic analyses demonstrated that all three STEC O91:H14 isolates were categorized into sequence type (ST) 33, of which two beef isolates were identical in their pulsotypes. Similar results were observed with two O121:H10 pork isolates (ST641; 88.2% similarity). Interestingly, 96.0% of the 100 human STEC isolates collected in Korea during 2003 to 2014 were serotyped as O91:H14, and the ST33 lineage was confirmed in approximately 72.2% (13/18 isolates) of human STEC O91:H14 isolates from diarrheal patients.

A Bioprocessed Polysaccharide from Lentinus edodes Mycelia Cultures with Turmeric Protects Chicks from a Lethal Challenge of Salmonella Gallinarum.

Our previous studies demonstrated that a bioprocessed polysaccharide (BPP) isolated from Lentinus edodes mushroom mycelia cultures supplemented with black rice bran can protect mice against Salmonella lipopolysaccharide-induced endotoxemia and reduce the mortality from Salmonella Typhimurium infection through upregulated T-helper 1 immunity. Here, we report that a BPP from L. edodes mushroom mycelia liquid cultures supplemented with turmeric (referred to as BPP-turmeric) alters chicken macrophage responses against avian-adapted Salmonella Gallinarum and protects chicks against a lethal challenge from Salmonella Gallinarum. In vitro analyses revealed that the water extract of BPP-turmeric (i) changed the protein expression or secretion profile of Salmonella Gallinarum, although it was not bactericidal, (ii) reduced the phagocytic activity of the chicken-derived macrophage cell line HD-11 when infected with Salmonella Gallinarum, and (iii) significantly activated the transcription expression of interleukin (IL)-1ß, IL-10, tumor necrosis factor α, and inducible nitric oxide synthase in response to various Salmonella infections, whereas it repressed that of IL-4, IL-6, interferon-ß, and interferon-γ. We also found that BPP-turmeric (0.1 g/kg of feed) as a feed additive provided significant protection to 1-day-old chicks infected with a lethal dose of Salmonella Gallinarum. Collectively, these results imply that BPP-turmeric contains biologically active component(s) that protect chicks against Salmonella Gallinarum infection, possibly by regulating macrophage immune responses. Further studies are needed to evaluate the potential efficacy of BPP-turmeric as a livestock feed additive for the preharvest control of fowl typhoid or foodborne salmonellosis.

Reduction of Nfia gene expression and subsequent target genes by binge alcohol in the fetal brain.

The objective of the present study was to investigate the changes in gene expression in the fetal brain (forebrain and hippocampus) caused by maternal binge alcohol consumption. Pregnant C57BL/6J mice were treated intragastrically with distilled phosphate-buffered saline (PBS) or ethanol (2.9 g/kg) from embryonic day (ED) 8-12. Microarray analysis revealed that a significant number of genes were altered at ED 18 in the developing brain. Specifically, in hippocampus, nuclear factor one alpha (Nfia) and three N-methyl-D-aspartate (Nmda) receptors (Nmdar1, Nmdar2b, and Nmdar2d) were down-regulated. The transcription factor Nfia controls gliogenesis, cell proliferation and Nmda-induced neuronal survival by regulating the expression of target genes. Some of the Nfia-target gene (Aldh1a, Folh1, Gjb6, Fgf1, Neurod1, Sept4, and Ntsr2) expressions were also altered as expected. These results suggest that the altered expression of Nfia and Nmda receptors may be associated with the etiology of fetal alcohol syndrome (FAS). The data presented in this report will contribute to the understanding of the molecular mechanisms associated with the effects of alcohol in FASD individuals.

Transcription-related element gene expression pattern differs between microglia and macrophages during inflammation.

OBJECTIVE AND DESIGN: Microglia and macrophages play an important role in the innate and adaptive immune systems. Although the resident location of these cells is different, their functions during the polarization response due to various stimuli are very similar. The present study aimed to analyze differences in microglial and macrophage gene expression during inflammation. METHODS: Mouse microglial BV-2 cells were exposed to LPS (10 ng/ml). The levels of gene expression were measured using real-time RT-PCR and whole transcriptome shotgun sequencing. RESULTS: The level of Jmjd3 gene expression in activated microglia showed a similar pattern to that of macrophages. In both cell types, genes associated with the inflammation response were generally increased whereas genes associated with metabolic and biosynthetic processes were decreased. However, the expression of transcription-related elements other than genes encoding histone modification enzymes showed a significantly different pattern between microglia and macrophages. CONCLUSION: Although the function and the gene expression levels of histone modification enzymes showed a similar pattern in microglia and macrophages during inflammation, the expression of transcription-related elements in both cell types showed a completely different pattern.

Effects of the monoamine oxidase inhibitors pargyline and tranylcypromine on cellular proliferation in human prostate cancer cells.

Chemotherapy is one of the therapeutic strategies that has been used for the inhibition of cancer cell proliferation in several types of cancer, including prostate cancer. Although monoamine oxidase (MAO) inhibitors, phytoestrogen and antioxidants used in chemotherapy have been systematically studied, their effects on cancer cell growth remain to be fully understood. The purpose of this study was to investigate the effects of the MAO inhibitors, pargyline and tranylcypromine on cell survival in human prostate carcinoma (LNCaP-LN3) cells. After treating LNCaP-LN3 cells with pargyline or tranylcypromine, we examined cell proliferation, cell cycle pattern, apoptosis and the expression levels of apoptosis-related genes. The proliferation of cells exposed to pargyline decreased in a dose- and time-dependent manner, while tranylcypromine-treated cells showed the opposite results. Treatment with pargyline significantly induced cell cycle arrest at the G1 phase compared to the control and tranylcypromine-treated cells. In addition, pargyline induced an increase in the cell death rate by promoting apoptosis; however, tranylcypromine had no effect on LNCaP-LN3 cells. Based on our results, we suggest that pargyline is more powerful than tranylcypromine for the treatment of human prostate cancer.

Proteomic changes induced by histone demethylase JMJD3 in TNF alpha-treated human monocytic (THP-1) cells.

JMJD3, a Jumonji C family histone demethylase, plays an important role in the regulation of inflammation induced by the transcription factor nuclear factor-kappa B (NF-κB) in response to various stimuli. JMJD3 is a histone-3 lysine-27 trimethylation (H3K27me3) demethylase, a histone mark associated with transcriptional repression and activation of a diverse set of genes. The present study assessed stable JMJD3 knockdown (KD)-dependent proteomic profiling in human leukemia monocyte (THP-1) cells to analyze the JMJD3-mediated differential changes of marker expression in inflammatory cells. To analyze the protein expression profile of tumor necrosis factor-alpha (TNF-α)-stimulated JMJD3-kd THP-1 cells, we employed matrix-assisted-laser-desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Additionally, Ingenuity Pathways Analysis (IPA) was applied to establish the molecular networks. A comparative proteomic profile was determined in TNF-α-treated both of JMJD3-kd THP-1 cells and THP-1 scrambled (sc) cells. The expression of tripartite motif protein (TRIM5), glutathione peroxidase (GPx), glia maturation factor-γ (GMFG), caspase recruitment domain family, member 14 (CARMA2), and dUTP pyrophosphatase were significantly down-regulated, whereas heat shock protein beta-1 (HspB1) and prohibition were significantly up-regulated in JMJD3-kd THP-1 cells. The molecular and signaling networks of the differentially expressed proteins in JMJD3-kd THP-1 cells were determined by IPA. The molecular network signatures and functional proteomics obtained in this study may facilitate the suppression of different key inflammatory regulators through JMJD3-attenuation, which would be crucial to evaluate potential therapeutic targets and to elucidate the molecular mechanism of JMJD3-kd dependent effects in THP-1 cells.

Effects of the activated mitogen-activated protein kinase pathway via the c-ros receptor tyrosine kinase on the T47D breast cancer cell line following alcohol exposure.

Compared to other cancers affecting women, breast cancer is significantly associated with alcohol consumption. However, the principles underlying the carcinogenesis of alcohol-induced breast cancer and the related metastatic mechanisms have yet to be established. To observe the effect of alcohol on the growth regulation in breast cancer cells, we identified differentially expressed proteins in alcohol-exposed human breast cancer T47D cells using gel-based proteomics analysis. The expression of c-ros receptor tyrosine kinase (ROS1) was increased and activated by autophosphorylation, thereby activating mitogen- and stress-activated protein kinase 1 (MSK1) through the mitogen­activated protein kinase (MAPK) pathway; activated MSK1, in turn, phosphorylated histone 3 serine 10 (H3S10p) residues in the nucleus. The increase in H3S10 phosphorylation consequently increased the level of expression of immediate-early gene such as c-fos. This study demonstrated that when breast cancer cells are exposed to alcohol, phosphorylated ROS1 activates MSK1 via Erk1/2 in the MAPK pathway, which then induces modifications to histone residues that regulate gene expression by 14-3-3 protein recruitment, leading to a lack of control of breast cancer cell proliferation.

Functional analysis of histone demethylase Jmjd2b on lipopolysaccharide-treated murine neural stem cells (NSCs).

Neural stem cell (NSC) neurogenesis is the formation of new neurons by which the brain maintains its lifelong plasticity in response to extrinsic and intrinsic changes. Here, we show the effect of lipopolysaccharides (LPS) as an in vitro model of inflammation on NSCs to determine whether the inflammatory mediators can epigenetically affect NSCs and alter their proliferation and differentiation abilities. To study the effect of LPS on NSCs, we used an immortalized mouse neuroectodermal stem cell line, NE-4C. We found that Jmjd2b, histone-3 lysine-9 di-/tri-methyl (H3K9me2/3) demethylase, is functional following LPS treatment and is crucial in multiple signaling pathways and biological processes. The global gene expression levels were detected in Jmjd2b-knockdown (kd) NE-4C cells and in LPS-stimulated Jmjd2b-kd NE-4C cells using an Affymetrix GeneChip(®) Mouse Gene 1.0 ST Array. In addition, the datasets were analyzed using MetaCore Pathway Analysis (GeneGo). The attenuation of Jmjd2b in NE-4C cells significantly affected the p65, iNOS, Bcl2, and TGF-ß expression levels and had downstream effects on related signaling pathways. In addition, chromatin immunoprecipitation revealed that Jmjd2b-kd could inhibit the Notch1, IL-1ß, and IL-2 genes by recruiting repressive H3K9me3 to their promoters. Moreover, this study highlights Jmjd2b role in LPS-mediated inflammation, which suggests an epigenetic regulation in NE-4C cells. Finally, this study establishes novel Jmjd2b targets that potentiate a biological rationale involving Jmjd2b in NSC inflammation.

Proteomic analysis of the copper ion-induced stress response in a human embryonic carcinoma cell line.

Excessive exposure to copper, a redox-active metal, generates free radicals, which can cause cellular damage. In this study, we aim to identify the proteins that are up- or downregulated by copper exposure in human embryonic carcinoma (NCCIT) cells and to understand the mechanisms that play a role in the copper-induced stress response. After exposure to copper ions, the cells showed upregulated levels of 78 kDa glucose-regulated protein, fibrillin 1, CWC22 spliceosome-associated protein (KIAA1604), heat shock protein (HSP) 60, and HSP70, while the tumor necrosis factor receptor-associated factor 6, vimentin, 14-3-3 protein zeta, and RAC-beta (AKT2) serine/threonine protein kinase were downregulated. The GeneGo Process Networks of the proteins upregulated by copper ions were analyzed, and the 3 highest-scoring networks from the proteins upregulated by copper ions are presented here. In particular, the increased level of HSP70 in response to copper ions occurred in a dose-dependent manner, indicating that HSP70 could be a potential biomarker for copper toxicity in mammalian cells.

Proteomic analysis of Terminalia chebula extract-dependent changes in human lymphoblastic T cell protein expression.

Terminalia chebula is a native plant from southern Asia to southwestern China that is used in traditional medicine for the treatment of malignant tumors and diabetes. This plant also has antibacterial and immunomodulatory properties. The present study assessed T. chebula extract-dependent protein expression changes in Jurkat cells. Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry and Ingenuity Pathways Analysis (IPA) were performed to assess protein expression and networks, respectively. A comparative proteomic profile was determined in T. chebula extract (50 µg/mL)-treated and control cells; the expressions of ß-tubulin, ring finger and CHY zinc finger domain containing 1, and insulin-like growth factor 1 receptor kinase were significantly down-regulated in T. chebula extract-treated Jurkat cells. Moreover, the molecular basis for the T. chebula extract-dependent protein expression changes in Jurkat cells was determined by IPA. Treatment with the T. chebula extract significantly inhibited nuclear factor-κB activity and affected the proteomic profile of Jurkat cells. The molecular network signatures and functional proteomics obtained in this study may facilitate the evaluation of potential antitumor therapeutic targets and elucidate the molecular mechanism of T. chebula extract-dependent effects in Jurkat cells.

Sodium arsenite dependent protein expression analysis on human embryonic carcinoma (NCCIT) cell line.

Due to their pluripotent nature, embryonic carcinoma (EC) cell lines are useful for the study of basic and applied aspects of medical therapeutics, disease management and environmental mutagenesis. We evaluated the teratogenic like effects of inorganic arsenic during the early stages of cellular development in NCCIT cells, a type of human EC cells. Using matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and MetaCore pathway analysis software (GeneGo), the proteomic expression profiles of NCCIT cells after either short- or long-term sodium arsenite (NaAsO(2)) treatment and of NCCIT cell-derived embryoid bodies (EBs) after short-term NaAsO(2) treatment were studied to determine the toxic effects on the process of normal growth and differentiation. The protein expression profiles of the NaAsO(2)-treated NCCIT cells and EBs were compared with that of untreated NCCIT cells. Short-term NaAsO(2) treatment resulted in the down-regulation of most proteins, with heat shock 90-kDa protein (HSP90) and valosin-containing protein (VCP) being significantly downregulated. Long-term NaAsO(2) treatment caused marked up-regulation of B23/nucleophosmin, glucose regulated protein 78-kDa (GRP78), unc-51-like kinase 3 (ULK3), toll-like receptor 6 precursor (TLR6) and mitogen-activated protein kinase 7 isoform A (MAP3K7). NaAsO(2) treatment of the NCCIT cell-derived EBs resulted in the down-regulation of several tumor-suppressor proteins. Taken together, these data suggest that a wide spectrum of cellular responses is induced to cope with chronic non-lethal toxic stresses in NCCIT cells.

Effects of acute ethanol treatment on NCCIT cells and NCCIT cell-derived embryoid bodies (EBs).

This study used human embryonic carcinoma (NCCIT) cells to evaluate genotoxicity and other effects of ethanol in earlier stages of cellular development. This undifferentiated pluripotent cell line has unlimited self-renewal capacity and has been shown to differentiate in vitro. We analyzed proteome expression profile of ethanol-treated NCCIT cells and NCCIT cell-derived embryoid bodies (EBs) by MALDI-TOF MS. To test the role of ethanol as an embryotoxic and/or teratogenic factor, MetaCore pathway analysis software (GeneGO) was used to evaluate the process of normal growth and differentiation of NCCIT cells and EBs. We compared the different protein expression profiles of ethanol-treated versus untreated NCCIT cells and NCCIT cell-derived EBs. The ethanol-treated NCCIT cells demonstrate significant up regulation of SMAP1, dual specificity phosphatase 1 and pro isomerase domain-containing 1, cytokeratin 18, triosephosphate isomerase and beta-tubulin. However, ethanol-treated NCCIT cell-derived EBs exhibited upregulated signatures of different proteins, including CDC25B phosphatase, alpha-enolase, 3-phosphoglycerate dehydrogenase and tumor suppressor patched L' isoform, which suggests that ethanol may play a different role in EBs. These proteins exert their function on transcriptional and translational processes. Moreover, the functional proteomic analysis confirms the relationship between ethanol and ethanol-regulated genes and various signaling pathways and networks. The data presented in this study contribute toward the understanding of the molecular mechanisms of ethanol in NCCIT cells and NCCIT cell-derived EBs.

Comparative proteome analysis of Bacillus anthracis with pXO1 plasmid content.

Bacillus anthracis the causative agent of anthrax, is an important pathogen among the Bacillus cereus group of species because of its physiological characteristics and its importance as a biological warfare agent. Tripartite anthrax toxin proteins and a poly-D-glutamic acid capsule are produced by B. anthracis vegetative cells during mammalian hosts infection and when cultured in conditions that are thought to mimic the host environment. To identify the factors regulating virulence in B. anthracis the whole cell proteins were extracted from two B. anthracis strains and separated by narrow range immobilized pH gradient (IPG) strips (pH 4-7), followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins that were differentially expressed were identified by the peptide fingerprinting using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). A total of 23 proteins were identified as being either upregulated or downregulated in the presence or absence of the virulence plasmid pXO1. Two plasmid encoded proteins and 12 cellular proteins were identified and documented as potential virulence factors.