ABSTRACT
Mammary gland-derived Escherichia coli is an important pathogen causing dairy cow mastitis. Mammary gland mucosal immunity against infectious E. coli mainly depends on recognition of pathogen-associated molecular patterns by innate receptors. Stimulator of interferon (IFN) gene (STING) has recently been the dominant mediator in reacting to bacterial intrusion and preventing inflammatory disorders. In this study, we first proved that the diguanylate cyclase YeaJ relieves mouse mammary gland pathological damage by changing E. coli phenotypic and host STING-dependent innate immunity responses. YeaJ decreases mammary gland circular vacuoles, bleeding, and degeneration in mice. In addition, YeaJ participates in STING-IRF3 signaling to regulate inflammation in vivo. In vitro, YeaJ decreases damage to macrophages (RAW264.7) but not to mouse mammary epithelial cells (EpH4-Ev). Consistent with the results in mouse mammary glands, YeaJ significantly activates the STING/TBK1/IRF3 pathway in RAW264.7 macrophages as well. In conclusion, the deletion of yeaJ facilitates E. coli NJ17 escape from STING-dependent innate immunity recognition in vitro and in vivo. This study highlights a novel role for YeaJ in E. coli infection, which provides a better understanding of host-bacterium interactions and potential prophylactic strategies for infections. IMPORTANCE E. coli is the etiological agent of environmental mastitis in dairy cows, which causes massive financial losses worldwide. However, the pathophysiological role of YeaJ in the interaction between E. coli and host remains unclear. We found that YeaJ significantly influences various biological characteristics and suppresses severe inflammatory response as well as greater damage. YeaJ alleviates damage to macrophages (RAW264.7) and mouse mammary gland. Moreover, these effects of YeaJ are achieved at least partial by mediating the STING-IRF3 signaling pathway. In conclusion, the deletion of yeaJ facilitates E. coli NJ17 escape from STING-dependent innate immunity recognition in vitro and in vivo. This study is the basis for further research to better understand host-bacterium interactions and provides potential prophylactic strategies for infections.
Subject(s)
Epithelial Cells/microbiology , Escherichia coli Proteins/metabolism , Escherichia coli/immunology , Escherichia coli/metabolism , Macrophages/microbiology , Phosphorus-Oxygen Lyases/metabolism , Animals , Biofilms/growth & development , Cell Adhesion , Escherichia coli Proteins/genetics , Female , Gene Expression Regulation, Bacterial/immunology , Mammary Glands, Animal/cytology , Mice , Movement , Mutation , Phosphorus-Oxygen Lyases/genetics , RAW 264.7 CellsABSTRACT
The proportion of Staphylococcus aureus in the skin microbiome is associated with the severity of inflammation in the skin disease atopic dermatitis. Staphylococcus epidermidis, a commensal skin bacterium, inhibits the growth of S. aureus in the skin. Therefore, the balance between S. epidermidis and S. aureus in the skin microbiome is important for maintaining healthy skin. In the present study, we demonstrated that the heat-treated culture supernatant of Delftia acidovorans, a member of the skin microbiome, inhibits the growth of S. epidermidis, but not that of S. aureus. Comprehensive gene expression analysis by RNA sequencing revealed that culture supernatant of D. acidovorans increased the expression of genes related to glycolysis and the tricarboxylic acid cycle (TCA) cycle in S. epidermidis. Malonate, an inhibitor of succinate dehydrogenase in the TCA cycle, suppressed the inhibitory effect of the heat-treated culture supernatant of D. acidovorans on the growth of S. epidermidis. Reactive oxygen species production in S. epidermidis was induced by the heat-treated culture supernatant of D. acidovorans and suppressed by malonate. Further, the inhibitory effect of the heat-treated culture supernatant of D. acidovorans on the growth of S. epidermidis was suppressed by N-acetyl-L-cysteine, a free radical scavenger. These findings suggest that heat-resistant substances secreted by D. acidovorans inhibit the growth of S. epidermidis by inducing the production of reactive oxygen species via the TCA cycle.
Subject(s)
Delftia acidovorans/immunology , Dermatitis, Atopic/immunology , Skin/microbiology , Staphylococcal Infections/immunology , Staphylococcus epidermidis/isolation & purification , Bacterial Proteins/genetics , Bacterial Proteins/immunology , Bacterial Proteins/metabolism , Citric Acid Cycle/immunology , Delftia acidovorans/genetics , Delftia acidovorans/metabolism , Dermatitis, Atopic/microbiology , Dermatitis, Atopic/pathology , Gene Expression Regulation, Bacterial/immunology , Humans , Microbiota/immunology , RNA-Seq , Reactive Oxygen Species/metabolism , Skin/immunology , Skin/pathology , Staphylococcal Infections/microbiology , Staphylococcal Infections/pathology , Staphylococcus aureus/immunology , Staphylococcus aureus/isolation & purification , Staphylococcus epidermidis/immunologyABSTRACT
Sensing and responding to environmental signals is critical for bacterial pathogens to successfully infect and persist within hosts. Many bacterial pathogens sense temperature as an indication they have entered a new host and must alter their virulence factor expression to evade immune detection. Using secondary structure prediction, we identified an RNA thermosensor (RNAT) in the 5' untranslated region (UTR) of tviA encoded by the typhoid fever-causing bacterium Salmonella enterica serovar Typhi (S. Typhi). Importantly, tviA is a transcriptional regulator of the critical virulence factors Vi capsule, flagellin, and type III secretion system-1 expression. By introducing point mutations to alter the mRNA secondary structure, we demonstrate that the 5' UTR of tviA contains a functional RNAT using in vitro expression, structure probing, and ribosome binding methods. Mutational inhibition of the RNAT in S. Typhi causes aberrant virulence factor expression, leading to enhanced innate immune responses during infection. In conclusion, we show that S. Typhi regulates virulence factor expression through an RNAT in the 5' UTR of tviA. Our findings demonstrate that limiting inflammation through RNAT-dependent regulation in response to host body temperature is important for S. Typhi's "stealthy" pathogenesis.
Subject(s)
Gene Expression Regulation, Bacterial/immunology , Host Microbial Interactions/immunology , Salmonella typhi/genetics , Temperature , Typhoid Fever/microbiology , Bacterial Proteins/metabolism , Humans , Immune Evasion/immunology , Salmonella typhi/immunology , Transcription Factors/immunology , Transcription Factors/metabolism , Virulence Factors/genetics , Virulence Factors/metabolismABSTRACT
Evolutionary arms races are broadly prevalent among organisms including bacteria, which have evolved defensive strategies against various attackers. A common microbial aggression mechanism is the type VI secretion system (T6SS), a contact-dependent bacterial weapon used to deliver toxic effector proteins into adjacent target cells. Sibling cells constitutively express immunity proteins that neutralize effectors. However, less is known about factors that protect non-sibling bacteria from T6SS attacks independently of cognate immunity proteins. In this study, we observe that human Escherichia coli commensal strains sensitive to T6SS attacks from Vibrio cholerae are protected when co-cultured with glucose. We confirm that glucose does not impair V. cholerae T6SS activity. Instead, we find that cells lacking the cAMP receptor protein (CRP), which regulates expression of hundreds of genes in response to glucose, survive significantly better against V. cholerae T6SS attacks even in the absence of glucose. Finally, we show that the glucose-mediated T6SS protection varies with different targets and killers. Our findings highlight the first example of an extracellular small molecule modulating a genetically controlled response for protection against T6SS attacks. This discovery may have major implications for microbial interactions during pathogen-host colonization and survival of bacteria in environmental communities.
Subject(s)
Bacterial Infections/immunology , Escherichia coli/immunology , Glucose/metabolism , Type VI Secretion Systems/toxicity , Vibrio cholerae/pathogenicity , Bacterial Infections/microbiology , Bacterial Proteins/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression Regulation, Bacterial/immunology , HumansABSTRACT
Clostridioides difficile is a bacterial pathogen that causes a range of clinical disease from mild to moderate diarrhea, pseudomembranous colitis, and toxic megacolon. Typically, C. difficile infections (CDIs) occur after antibiotic treatment, which alters the gut microbiota, decreasing colonization resistance against C. difficile. Disease is mediated by two large toxins and the expression of their genes is induced upon nutrient depletion via the alternative sigma factor TcdR. Here, we use tcdR mutants in two strains of C. difficile and omics to investigate how toxin-induced inflammation alters C. difficile metabolism, tissue gene expression and the gut microbiota, and to determine how inflammation by the host may be beneficial to C. difficile. We show that C. difficile metabolism is significantly different in the face of inflammation, with changes in many carbohydrate and amino acid uptake and utilization pathways. Host gene expression signatures suggest that degradation of collagen and other components of the extracellular matrix by matrix metalloproteinases is a major source of peptides and amino acids that supports C. difficile growth in vivo. Lastly, the inflammation induced by C. difficile toxin activity alters the gut microbiota, excluding members from the genus Bacteroides that are able to utilize the same essential nutrients released from collagen degradation.
Subject(s)
Bacterial Proteins/metabolism , Bacterial Toxins/metabolism , Clostridioides difficile/metabolism , Clostridium Infections/immunology , Gastrointestinal Microbiome/immunology , Sigma Factor/metabolism , Animals , Anti-Bacterial Agents/adverse effects , Bacterial Proteins/genetics , Bacterial Toxins/genetics , Bacterial Toxins/immunology , Bacteroides/drug effects , Bacteroides/metabolism , Clostridioides difficile/genetics , Clostridioides difficile/immunology , Clostridium Infections/microbiology , Clostridium Infections/pathology , Disease Models, Animal , Extracellular Matrix/metabolism , Female , Gastrointestinal Microbiome/drug effects , Gene Expression Regulation, Bacterial/immunology , Host-Pathogen Interactions/genetics , Host-Pathogen Interactions/immunology , Humans , Intestinal Mucosa/immunology , Intestinal Mucosa/microbiology , Intestinal Mucosa/pathology , Male , Matrix Metalloproteinases/metabolism , Mice , Nutrients/metabolism , Proteolysis , RNA, Bacterial/genetics , RNA, Bacterial/isolation & purification , RNA-Seq , Sigma Factor/genetics , Sigma Factor/immunology , Transcriptome/immunologyABSTRACT
Mucus is a densely populated ecological niche that coats all non-keratinized epithelia, and plays a critical role in protecting the human body from infections. Although traditionally viewed as a physical barrier, emerging evidence suggests that mucus can directly suppress virulence-associated traits in opportunistic pathogens including Pseudomonas aeruginosa. However, the molecular mechanisms by which mucus affords this protection are unclear. Here, we show that mucins, and particularly their associated glycans, signal through the Dismed2 domain of the sensor kinase RetS in P. aeruginosa. We find that this RetS-dependent signaling leads to the direct inhibition of the GacS-GacA two-component system, the activity of which is associated with a chronic infection state. This signaling includes downregulation of the type VI secretion system (T6SS), and prevents T6SS-dependent bacterial killing by P. aeruginosa. Overall, these results shed light on how mucus impacts P. aeruginosa behavior, and may inspire novel approaches for controlling P. aeruginosa infections.
Subject(s)
Bacterial Proteins/metabolism , Mucin 5AC/metabolism , Polysaccharides/metabolism , Pseudomonas Infections/immunology , Pseudomonas aeruginosa/immunology , Animals , Bacterial Proteins/genetics , Disease Models, Animal , Down-Regulation , Gastric Mucosa/immunology , Gastric Mucosa/metabolism , Gastric Mucosa/microbiology , Gene Expression Regulation, Bacterial/immunology , Host-Pathogen Interactions/immunology , Humans , Pseudomonas Infections/microbiology , Pseudomonas aeruginosa/genetics , Pseudomonas aeruginosa/metabolism , Pseudomonas aeruginosa/pathogenicity , RNA-Seq , Swine , Transcription Factors/genetics , Transcription Factors/metabolism , Type VI Secretion Systems/genetics , Type VI Secretion Systems/metabolism , Virulence/geneticsABSTRACT
Unlike pathogens, which attack the host, commensal bacteria create a state of friendly coexistence. Here, we identified a mechanism of bacterial adaptation to the host niche, where they reside. Asymptomatic carrier strains were shown to inhibit RNA polymerase II (Pol II) in host cells by targeting Ser2 phosphorylation, a step required for productive mRNA elongation. Assisted by a rare, spontaneous loss-of-function mutant from a human carrier, the bacterial NlpD protein was identified as a Pol II inhibitor. After internalization by host cells, NlpD was shown to target constituents of the Pol II phosphorylation complex (RPB1 and PAF1C), attenuating host gene expression. Therapeutic efficacy of a recombinant NlpD protein was demonstrated in a urinary tract infection model, by reduced tissue pathology, accelerated bacterial clearance, and attenuated Pol II-dependent gene expression. The findings suggest an intriguing, evolutionarily conserved mechanism for bacterial modulation of host gene expression, with a remarkable therapeutic potential.
Subject(s)
Escherichia coli Infections , Escherichia coli Proteins , Escherichia coli , Gene Expression Regulation, Bacterial/immunology , Lipoproteins , RNA Polymerase II , Urinary Tract Infections , Animals , Cell Line, Tumor , Escherichia coli/genetics , Escherichia coli/immunology , Escherichia coli Infections/genetics , Escherichia coli Infections/immunology , Escherichia coli Proteins/genetics , Escherichia coli Proteins/immunology , Female , Humans , Lipoproteins/genetics , Lipoproteins/immunology , Mice , RNA Polymerase II/genetics , RNA Polymerase II/immunology , Urinary Tract Infections/genetics , Urinary Tract Infections/immunologyABSTRACT
Organelle remodeling is critical for cellular homeostasis, but host factors that control organelle function during microbial infection remain largely uncharacterized. Here, a genome-scale CRISPR/Cas9 screen in intestinal epithelial cells with the prototypical intracellular bacterial pathogen Salmonella led us to discover that type I IFN (IFN-I) remodels lysosomes. Even in the absence of infection, IFN-I signaling modified the localization, acidification, protease activity, and proteomic profile of lysosomes. Proteomic and genetic analyses revealed that multiple IFN-I-stimulated genes including IFITM3, SLC15A3, and CNP contribute to lysosome acidification. IFN-I-dependent lysosome acidification was associated with elevated intracellular Salmonella virulence gene expression, rupture of the Salmonella-containing vacuole, and host cell death. Moreover, IFN-I signaling promoted in vivo Salmonella pathogenesis in the intestinal epithelium where Salmonella initiates infection, indicating that IFN-I signaling can modify innate defense in the epithelial compartment. We propose that IFN-I control of lysosome function broadly impacts host defense against diverse viral and microbial pathogens.
Subject(s)
Epithelial Cells/immunology , Interferon Type I/metabolism , Intestinal Mucosa/immunology , Lysosomes/metabolism , Salmonella Infections/immunology , Animals , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , CRISPR-Cas Systems/genetics , Disease Models, Animal , Epithelial Cells/chemistry , Epithelial Cells/cytology , Epithelial Cells/metabolism , Gene Expression Regulation, Bacterial/immunology , HT29 Cells , Host-Pathogen Interactions/genetics , Host-Pathogen Interactions/immunology , Humans , Hydrogen-Ion Concentration , Immunity, Innate , Intestinal Mucosa/cytology , Intestinal Mucosa/microbiology , Lysosomes/chemistry , Lysosomes/immunology , Mice , Mice, Knockout , Necroptosis/immunology , Peptide Hydrolases/metabolism , Proteomics , Receptor, Interferon alpha-beta/genetics , Receptor, Interferon alpha-beta/metabolism , Salmonella Infections/microbiology , Salmonella typhimurium/immunology , Salmonella typhimurium/pathogenicity , Signal Transduction/immunology , Virulence/immunology , Virulence Factors/genetics , Virulence Factors/metabolismABSTRACT
Helicobacter pylori CagA protein plays an important role in the severity of the gastric diseases. Our aims were to clone the cagA 5'- conserved region of the gene, characterize the recombinant CagA (rCagA) protein by monoclonal antibodies (mAbs) and to use this protein for the detection of anti-CagA antibodies by an ELISA test. Our developed rCagA protein (67 kDa) showed an amino acid sequence homology of 83% and 80% with Western and East Asian type strains respectively. Two anti-rCagA (BS-53, CK-02) mAbs and 2 additional rCagA proteins of smaller sizes (60 kDa, 28 kDa) were developed for epitope determination. The BS-53 mAb recognized all 3 rCagA proteins while CK-02 mAb recognized only 2 of them indicating recognition of different epitopes. An in-house indirect ELISA using rCagA was developed to detect anti-CagA antibodies in sera of 59 patients. The ELISA results obtained when compared to those of the PCR gave a sensitivity, specificity and accuracy of 81%, 100% and 88% respectively. We have developed for the first time: a rCagA protein that showed high sequence homology with both Western and East Asian type strains and an indirect ELISA of high performance which can be used to detect anti-CagA antibodies in sera of infected patients worldwide.
Subject(s)
Antibodies, Monoclonal/immunology , Antigens, Bacterial/immunology , Bacterial Proteins/immunology , Gene Expression Regulation, Bacterial/immunology , Helicobacter Infections/immunology , Helicobacter pylori/immunology , Recombinant Proteins/immunology , Antibodies, Monoclonal/metabolism , Antigens, Bacterial/genetics , Antigens, Bacterial/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Blotting, Western , Cloning, Molecular , Enzyme-Linked Immunosorbent Assay , Helicobacter Infections/diagnosis , Helicobacter Infections/microbiology , Helicobacter pylori/genetics , Helicobacter pylori/metabolism , Humans , ROC Curve , Recombinant Proteins/metabolismABSTRACT
BACKGROUND: Mastitis is the most common disease in dairy cattle and the costliest for the dairy farming industry, as it lowers milk yield and quality. Mastitis occurs as a result of interactions between microorganisms and the individual genetic predispositions of each animal. Thus, it is important to fully understand the mechanisms underlying these interactions. Elucidating the immune response mechanisms can determine which genetic background makes an animal highly resistant to mastitis. We analyzed the innate immune responses of dairy cows naturally infected with coagulase-positive staphylococci (CoPS; N = 8) or coagulase-negative staphylococci (CoNS; N = 7), causing persistent mastitis (after several failed treatments) vs. infection-free (i.e., healthy [H]; N = 8) dairy cows. The expressions of the acute phase protein genes serum amyloid A3 (SAA3), haptoglobin (HP), ceruloplasmin (CP) genes in the tissues most exposed to pathogens- mammary gland cistern lining epithelial cells (CLECs) and mammary epithelial cells (MECs)-were analyzed. RESULTS: We found constitutive and extrahepatic expressions of the studied genes in both tissue types. HP expression in the MECs of the CoPS-infected group was higher than in the H group (p ≤ 0.05). Moreover, higher SAA3 expression in the CoPS and CoNS groups than in the H group (p = 0.06 and 0.08, respectively) was found. No differences between SAA3 and HP in CLECs were revealed, regardless of the pathogen type. However, higher expression of CP (p ≤ 0.05) in the CoPS group than in the H group was noted. CONCLUSIONS: The expressions of selected acute phase proteins were similar between CLECs and MECs, which means that CLECs are not only a mechanical barrier but are also responsible for the biological immune response. Our findings agree with the results of other authors describing the immunological response of MECs during chronic mastitis, but the results for CLECs are novel.
Subject(s)
Acute-Phase Proteins/metabolism , Gene Expression Regulation, Bacterial/immunology , Mammary Glands, Animal/metabolism , Mastitis, Bovine/metabolism , Staphylococcal Infections/veterinary , Acute-Phase Proteins/genetics , Animals , Cattle , Chronic Disease , Epithelium/metabolism , Female , Mastitis, Bovine/microbiology , Staphylococcal Infections/metabolism , Staphylococcal Infections/microbiologyABSTRACT
Studying emerging or neglected pathogens is often challenging due to insufficient information and absence of genetic tools. Dual RNA-seq provides insights into host-pathogen interactions, and is particularly informative for intracellular organisms. Here we apply dual RNA-seq to Orientia tsutsugamushi (Ot), an obligate intracellular bacterium that causes the vector-borne human disease scrub typhus. Half the Ot genome is composed of repetitive DNA, and there is minimal collinearity in gene order between strains. Integrating RNA-seq, comparative genomics, proteomics, and machine learning to study the transcriptional architecture of Ot, we find evidence for wide-spread post-transcriptional antisense regulation. Comparing the host response to two clinical isolates, we identify distinct immune response networks for each strain, leading to predictions of relative virulence that are validated in a mouse infection model. Thus, dual RNA-seq can provide insight into the biology and host-pathogen interactions of a poorly characterized and genetically intractable organism such as Ot.
Subject(s)
Gene Expression Regulation, Bacterial/immunology , Host-Pathogen Interactions/immunology , Neglected Diseases/immunology , Orientia tsutsugamushi/genetics , Scrub Typhus/immunology , Animals , Bacterial Proteins/genetics , Bacterial Proteins/immunology , Bacterial Proteins/metabolism , Cell Line , Disease Models, Animal , Feasibility Studies , Female , Genome, Bacterial , Human Umbilical Vein Endothelial Cells , Humans , Interferon Type I/immunology , Interferon Type I/metabolism , Interspersed Repetitive Sequences/genetics , Mice , Neglected Diseases/microbiology , Orientia tsutsugamushi/immunology , Orientia tsutsugamushi/pathogenicity , Proteomics , RNA, Bacterial/genetics , RNA, Bacterial/isolation & purification , RNA, Bacterial/metabolism , RNA, Untranslated/genetics , RNA, Untranslated/metabolism , RNA-Seq , Scrub Typhus/microbiology , Transcription, Genetic , Exome SequencingABSTRACT
The serum complement system is a first line of defense against bacterial invaders. Resistance to killing by serum enhances the capacity of Klebsiella pneumoniae to cause infection, but it is an incompletely understood virulence trait. Identifying and characterizing the factors responsible for preventing activation of, and killing by, serum complement could inform new approaches to treatment of K. pneumoniae infections. Here, we used functional genomic profiling to define the genetic basis of complement resistance in four diverse serum-resistant K. pneumoniae strains (NTUH-K2044, B5055, ATCC 43816, and RH201207), and explored their recognition by key complement components. More than 90 genes contributed to resistance in one or more strains, but only three, rfaH, lpp, and arnD, were common to all four strains. Deletion of the antiterminator rfaH, which controls the expression of capsule and O side chains, resulted in dramatic complement resistance reductions in all strains. The murein lipoprotein gene lpp promoted capsule retention through a mechanism dependent on its C-terminal lysine residue; its deletion led to modest reductions in complement resistance. Binding experiments with the complement components C3b and C5b-9 showed that the underlying mechanism of evasion varied in the four strains: B5055 and NTUH-K2044 appeared to bypass recognition by complement entirely, while ATCC 43816 and RH201207 were able to resist killing despite being associated with substantial levels of C5b-9. All rfaH and lpp mutants bound C3b and C5b-9 in large quantities. Our findings show that, even among this small selection of isolates, K. pneumoniae adopts differing mechanisms and utilizes distinct gene sets to avoid complement attack.
Subject(s)
Bacterial Outer Membrane Proteins/immunology , Carboxy-Lyases/immunology , Gene Expression Regulation, Bacterial/immunology , Genes, Bacterial , Immune Evasion , Klebsiella pneumoniae/immunology , Peptide Elongation Factors/immunology , Bacterial Outer Membrane Proteins/genetics , Blood Bactericidal Activity/immunology , Carboxy-Lyases/deficiency , Carboxy-Lyases/genetics , Complement C3b/genetics , Complement C3b/immunology , Complement Membrane Attack Complex/genetics , Complement Membrane Attack Complex/immunology , DNA Transposable Elements , Gene Expression Profiling , Gene Library , Humans , Klebsiella Infections/immunology , Klebsiella Infections/microbiology , Klebsiella pneumoniae/genetics , Klebsiella pneumoniae/pathogenicity , Mutation , Peptide Elongation Factors/deficiency , Peptide Elongation Factors/genetics , Sequence Analysis, DNAABSTRACT
Group A Streptococcus (GAS) is a human-specific pathogen that evades the host immune response through the elaboration of multiple virulence factors. Although many of these factors have been studied, numerous proteins encoded by the GAS genome are of unknown function. Herein, we characterize a biomimetic red blood cell (RBC)-captured protein of unknown function-annotated subsequently as S protein-in GAS pathophysiology. S protein maintains the hydrophobic properties of GAS, and its absence reduces survival in human blood. S protein facilitates GAS coating with lysed RBCs to promote molecular mimicry, which increases virulence in vitro and in vivo. Proteomic profiling reveals that the removal of S protein from GAS alters cellular and extracellular protein landscapes and is accompanied by a decrease in the abundance of several key GAS virulence determinants. In vivo, the absence of S protein results in a striking attenuation of virulence and promotes a robust immune response and immunological memory.
Subject(s)
Erythrocytes/immunology , Immune Evasion/immunology , Streptococcal Infections/immunology , Streptococcus/immunology , Animals , Bacterial Proteins/immunology , Cell Line , Gene Expression Profiling/methods , Gene Expression Regulation, Bacterial/immunology , Host-Pathogen Interactions/immunology , Humans , Mice , Mice, Inbred C57BL , Mice, Inbred ICR , Proteomics/methods , THP-1 Cells , Virulence/immunology , Virulence Factors/immunologyABSTRACT
Pseudomonas aeruginosa, a significant opportunistic pathogen, can participate in inter-species communication through signaling by cis-2-unsaturated fatty acids of the diffusible signal factor (DSF) family. Sensing these signals leads to altered biofilm formation and increased tolerance to various antibiotics, and requires the histidine kinase PA1396. Here, we show that the membrane-associated sensory input domain of PA1396 has five transmembrane helices, two of which are required for DSF sensing. DSF binding is associated with enhanced auto-phosphorylation of PA1396 incorporated into liposomes. Further, we examined the ability of synthetic DSF analogues to modulate or inhibit PA1396 activity. Several of these analogues block the ability of DSF to trigger auto-phosphorylation and gene expression, whereas others act as inverse agonists reducing biofilm formation and antibiotic tolerance, both in vitro and in murine infection models. These analogues may thus represent lead compounds to develop novel adjuvants improving the efficacy of existing antibiotics.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Proteins/metabolism , Fatty Acids, Unsaturated/metabolism , Histidine Kinase/metabolism , Pseudomonas Infections/drug therapy , Pseudomonas aeruginosa/physiology , Animals , Anti-Bacterial Agents/therapeutic use , Bacterial Proteins/genetics , Biofilms/drug effects , Disease Models, Animal , Drug Resistance, Bacterial , Female , Gene Expression Regulation, Bacterial/drug effects , Gene Expression Regulation, Bacterial/immunology , Histidine Kinase/genetics , Humans , Liposomes/metabolism , Mice , Mice, Inbred C57BL , Microbial Sensitivity Tests , Mutagenesis , Phosphorylation , Polymyxins/pharmacology , Polymyxins/therapeutic use , Pseudomonas Infections/immunology , Pseudomonas Infections/microbiology , Virulence Factors/genetics , Virulence Factors/metabolismABSTRACT
Gram-positive bacteria process and release small peptides, or pheromones, that act as signals for the induction of adaptive traits, including those involved in pathogenesis. One class of small signaling pheromones is the cyclic autoinducing peptides (AIPs), which regulate expression of genes that orchestrate virulence and persistence in a range of microbes, including staphylococci, listeriae, clostridia, and enterococci. In a genetic screen for Staphylococcus aureus secreted virulence factors, we identified an S. aureus mutant containing an insertion in the gene SAUSA300_1984 (mroQ), which encodes a putative membrane-embedded metalloprotease. A ΔmroQ mutant exhibited impaired induction of Toll-like receptor 2-dependent inflammatory responses from macrophages but elicited greater production of the inflammatory cytokine interleukin-1ß and was attenuated in a murine skin and soft tissue infection model. The ΔmroQ mutant phenocopies an S. aureus mutant containing a deletion of the accessory gene regulatory system (Agr), wherein both strains have significantly reduced production of secreted toxins and virulence factors but increased surface protein A abundance. The Agr system controls virulence factor gene expression in S. aureus by sensing the accumulation of AIP via the histidine kinase AgrC and the response regulator AgrA. We provide evidence to suggest that MroQ acts within the Agr pathway to facilitate the optimal processing or export of AIP for signal amplification through AgrC/A and induction of virulence factor gene expression. Mutation of MroQ active-site residues significantly reduces AIP signaling and attenuates virulence. Altogether, this work identifies a new component of the Agr quorum-sensing circuit that is critical for the production of S. aureus virulence factors.
Subject(s)
Bacterial Proteins/immunology , Membrane Proteins/immunology , Peptide Hydrolases/immunology , Quorum Sensing/immunology , Staphylococcal Infections/prevention & control , Staphylococcus aureus/immunology , Virulence/immunology , Gene Expression Regulation, Bacterial/immunologyABSTRACT
Numerous factors have, to date, been identified as playing a role in the regulation of Agr activity in Staphylococcus aureus, including transcription factors, antisense RNAs, and host elements. Herein we investigated the product of SAUSA300_1984 (termed MroQ), a transmembrane Abi-domain/M79 protease-family protein, as a novel effector of this system. Using a USA300 mroQ mutant, we observed a drastic reduction in proteolysis, hemolysis, and pigmentation that was fully complementable. This appears to result from diminished agr activity, as transcriptional analysis revealed significant decreases in expression of both RNAII and RNAIII in the mroQ mutant. Such effects appear to be direct, rather than indirect, as known agr effectors demonstrated limited alterations in their activity upon mroQ disruption. A comparison of RNA sequencing data sets for both mroQ and agr mutants revealed a profound overlap in their regulomes, with the majority of factors affected being known virulence determinants. Importantly, the preponderance of alterations in expression were more striking in the agr mutant, indicating that MroQ is necessary, but not sufficient, for Agr function. Mechanism profiling revealed that putative residues for metalloprotease activity within MroQ are required for its Agr-controlling effect; however, this was not wielded at the level of AgrD processing. Virulence assessment demonstrated that both mroQ and agr mutants exhibited increased formation of renal abscesses but decreased skin abscess formation alongside diminished dermonecrosis. Collectively, we present the characterization of a novel agr effector in S. aureus which would appear to be a direct regulator, potentially functioning via interaction with the AgrC histidine kinase.
Subject(s)
Bacterial Proteins/immunology , Gene Expression Regulation, Bacterial/immunology , Staphylococcal Infections/immunology , Staphylococcal Infections/pathology , Staphylococcus aureus/immunology , Transcription Factors/immunology , Virulence Factors/immunology , Animals , Bacterial Proteins/genetics , Female , Gene Expression Regulation, Bacterial/genetics , Humans , Mice , Models, Animal , Staphylococcal Infections/genetics , Staphylococcus aureus/genetics , Transcription Factors/genetics , Virulence Factors/geneticsABSTRACT
Incidence of whooping cough (pertussis), a bacterial infection of the respiratory tract caused by the bacterium Bordetella pertussis, has reached levels not seen since the 1950s. Antibiotics fail to improve the course of disease unless administered early in infection. Therefore, there is an urgent need for the development of antipertussis therapeutics. Sphingosine-1-phosphate receptor (S1PR) agonists have been shown to reduce pulmonary inflammation during Bordetella pertussis infection in mouse models. However, the mechanisms by which S1PR agonists attenuate pertussis disease are unknown. We report the results of a transcriptome sequencing study examining pulmonary transcriptional responses in B. pertussis-infected mice treated with S1PR agonist AAL-R or vehicle control. This study identified peptidoglycan recognition protein 4 (PGLYRP4) as one of the most highly upregulated genes in the lungs of infected mice following S1PR agonism. PGLYRP4, a secreted, innate mediator of host defenses, was found to limit early inflammatory pathology in knockout mouse studies. Further, S1PR agonist AAL-R failed to attenuate pertussis disease in PGLYRP4 knockout (KO) mice. B. pertussis virulence factor tracheal cytotoxin (TCT), a secreted peptidoglycan breakdown product, induces host tissue damage. TCT-oversecreting strains were found to drive an early inflammatory response similar to that observed in PGLYRP4 KO mice. Further, TCT-oversecreting strains induced significantly greater pathology in PGLYRP4-deficient animals than their wild-type counterparts. Together, these data indicate that S1PR agonist-mediated protection against pertussis disease is PGLYRP4 dependent. Our data suggest PGLYRP4 functions, in part, by preventing TCT-induced airway damage.
Subject(s)
Bordetella pertussis/immunology , Carrier Proteins/metabolism , Receptors, Lysosphingolipid/agonists , Whooping Cough/immunology , Animals , Disease Models, Animal , Gene Expression Regulation, Bacterial/immunology , Mice , Mice, Inbred C57BL , Mice, KnockoutABSTRACT
The nosocomial pathogen Acinetobacter baumannii is a significant threat due to its ability to cause infections refractory to a broad range of antibiotic treatments. We show here that a highly conserved sensory-transduction system, BfmRS, mediates the coordinate development of both enhanced virulence and resistance in this microorganism. Hyperactive alleles of BfmRS conferred increased protection from serum complement killing and allowed lethal systemic disease in mice. BfmRS also augmented resistance and tolerance against an expansive set of antibiotics, including dramatic protection from ß-lactam toxicity. Through transcriptome profiling, we showed that BfmRS governs these phenotypes through global transcriptional regulation of a post-exponential-phase-like program of gene expression, a key feature of which is modulation of envelope biogenesis and defense pathways. BfmRS activity defended against cell-wall lesions through both ß-lactamase-dependent and -independent mechanisms, with the latter being connected to control of lytic transglycosylase production and proper coordination of morphogenesis and division. In addition, hypersensitivity of bfmRS knockouts could be suppressed by unlinked mutations restoring a short, rod cell morphology, indicating that regulation of drug resistance, pathogenicity, and envelope morphogenesis are intimately linked by this central regulatory system in A. baumannii. This work demonstrates that BfmRS controls a global regulatory network coupling cellular physiology to the ability to cause invasive, drug-resistant infections.
Subject(s)
Acinetobacter baumannii/genetics , Acinetobacter baumannii/metabolism , Drug Resistance, Bacterial/genetics , Acinetobacter Infections/pathology , Alleles , Animals , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/metabolism , Biofilms/growth & development , Drug Resistance, Bacterial/immunology , Drug Resistance, Bacterial/physiology , Drug Resistance, Multiple, Bacterial/genetics , Female , Gene Expression Regulation, Bacterial/genetics , Gene Expression Regulation, Bacterial/immunology , Homeostasis/drug effects , Mice , Mice, Inbred C57BL , Microbial Sensitivity Tests , Signal Transduction/drug effects , Transcriptome/genetics , Transcriptome/immunology , Virulence/drug effects , Virulence/immunology , beta-Lactam Resistance/genetics , beta-Lactamases/metabolismABSTRACT
Many bacterial pathogens coordinately regulate genes encoding important metabolic pathways during disease progression, including the phosphoenolpyruvate (PEP)-phosphotransferase system (PTS) for uptake of carbohydrates. The Gram-positive Group A Streptococcus (GAS) is a pathogen that infects multiple tissues in the human host. The virulence regulator Mga in GAS can be phosphorylated by the PTS, affecting Mga activity based on carbohydrate availability. Here, we explored the effects of glucose availability on the Mga regulon. RNA-seq was used to identify transcriptomic differences between the Mga regulon grown to late log phase in the presence of glucose (THY) or after glucose has been expended (C media). Our results revealed a correlation between the genes activated in C media with those known to be repressed by CcpA, indicating that C media mimics a non-preferred sugar environment. Interestingly, we found very little overlap in the Mga regulon from GAS grown in THY versus C media beyond the core virulence genes. We also observed an alteration in the phosphorylation status of Mga, indicating that the observed media differences in the Mga regulon may be directly attributed to glucose levels. Thus, these results support an in vivo link between glucose availability and virulence regulation in GAS.
Subject(s)
Blood Glucose/immunology , Gene Expression Regulation, Bacterial/immunology , Regulon/immunology , Streptococcal Infections/immunology , Streptococcus pyogenes/pathogenicity , Bacterial Proteins/genetics , Bacterial Proteins/immunology , Bacterial Proteins/metabolism , Blood Glucose/metabolism , Gene Expression Profiling , Gene Expression Regulation, Bacterial/genetics , Humans , Phosphotransferases , Sequence Analysis, RNA , Streptococcal Infections/blood , Streptococcal Infections/microbiology , Streptococcus pyogenes/genetics , Streptococcus pyogenes/immunology , Virulence/genetics , Virulence/immunology , Virulence Factors/genetics , Virulence Factors/immunology , Virulence Factors/metabolismABSTRACT
Background: Staphylococcus aureus and Pseudomonas aeruginosa are key bacterial pathogens of the respiratory tract in patients with cystic fibrosis (CF). Although P. aeruginosa chronic bronchial infection is associated with a poorer prognosis, the consequences of S. aureus colonization on CF outcomes are controversial. Methods: In this paper, murine models of infection resembling traits of the CF human airways disease have been revisited using an infection schedule that mimics the sequence of events of pulmonary disease in CF patients. First, mice were infected with S. aureus, embedded in agar beads; this was followed by P. aeruginosa infection and analysis of bacterial load, leukocyte infiltration, and lung tissue damage. Results: We reveal that (1) S. aureus promotes severe lesions including abscess formation, (2) S. aureus increases the risk of subsequent chronic P. aeruginosa respiratory infection, and (3) once the chronic infection has been established, P. aeruginosa influences most of the inflammatory responses independent of S. aureus. Conclusions: Our findings established the significance of S. aureus colonization per se and the impact on the subsequent P. aeruginosa infection. This would point towards a thorough assessment for the need of treatment against S. aureus.