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The Candidate Phyla Radiation (CPR) encompasses widespread uncultivated bacteria with reduced genomes and limited metabolic capacities. Most CPR bacteria lack the minimal set of enzymes required for peptidoglycan (PG) synthesis, leaving it unclear how these bacteria produce this essential envelope component. In this study, we analysed the distribution of d-amino acid racemases that produce the universal PG components d-glutamate (d-Glu) or d-alanine (d-Ala). We also examined moonlighting enzymes that synthesize d-Glu or d-Ala. Unlike other phyla in the domain Bacteria, CPR bacteria do not exhibit these moonlighting activities and have, at most, one gene encoding either a Glu or Ala racemase. One of these 'orphan' racemases is a predicted Glu racemase (MurICPR) from the CPR bacterium Candidatus Saccharimonas aalborgenesis. The expression of MurICPR restores the growth of a Salmonella d-Glu auxotroph lacking its endogenous racemase and results in the substitution of l-Ala by serine as the first residue in a fraction of the PG stem peptides. In vitro, MurICPR exclusively racemizes Glu as a substrate. Therefore, Ca. Saccharimonas aalborgensis may couple Glu racemization to serine and d-Glu incorporation into the stem peptide. Our findings provide the first insights into the synthesis of PG by an uncultivated environmental bacterium and illustrate how to experimentally test enzymatic activities from CPR bacteria related to PG metabolism.
Assuntos
Isomerases de Aminoácido , Isomerases de Aminoácido/genética , Isomerases de Aminoácido/química , Isomerases de Aminoácido/metabolismo , Racemases e Epimerases , Bactérias/metabolismo , Ácido Glutâmico/metabolismo , SerinaRESUMO
Listeria monocytogenes, a contaminant of raw milk, includes hypervirulent clonal complexes (CC) like CC1, CC4, and CC6, highly overrepresented in dairy products when compared to other food types. Whether their higher prevalence in dairy products is the consequence of a growth advantage in this food remains unknown. We examined growth kinetics of five L. monocytogenes isolates (CC1, CC4, CC6, CC9, and CC121) at 37 and 4 °C in ultra-high temperature (UHT) milk and raw milk. At 4 °C, hypovirulent CC9 and CC121 isolates exhibit better growth parameters in UHT milk compared to the hypervirulent CC1, CC4, and CC6 isolates. CC9 isolate in raw milk at 4 °C exhibited the fastest growth and the highest final concentrations. In contrast, hypervirulent isolates (CC1, CC4, and CC6) displayed better growth rates in UHT milk at 37 °C, the mammalian host temperature. Proteomic analysis of representative hyper- (CC1) and hypovirulent (CC9) isolates showed that they respond to milk cues differently with CC-specific traits. Proteins related to metabolism (such as LysA or different phosphotransferase systems), and stress response were upregulated in both isolates during growth in UHT milk. Our results show that there is a Listeria CC-specific and a Listeria CC-common response to the milk environment. These findings shed light on the overrepresentation of hypervirulent L. monocytogenes isolates in dairy products, suggesting that CC1 and CC4 overrepresentation in dairy products made of raw milk may arise from contamination during or after milking at the farm and discard an advantage of hypervirulent isolates in milk products when stored at refrigeration temperatures.
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The general stress response (GSR) in Listeria monocytogenes plays a critical role in the survival of this pathogen in the host gastrointestinal tract. The GSR is regulated by the alternative sigma factor B (σB), whose role in protection against acid stress is well established. Here, we investigated the involvement of the stressosome, a sensory hub, in transducing low pH signals to induce the GSR. Mild acid shock (15 min at pH 5.0) activated σB and conferred protection against a subsequent lethal pH challenge. A mutant strain where the stressosome subunit RsbR1 was solely present retained the ability to induce σB activity at pH 5.0. The role of stressosome phosphorylation in signal transduction was investigated by mutating the putative phosphorylation sites in the core stressosome proteins RsbR1 (rsbR1-T175A, -T209A, -T241A) and RsbS (rsbS-S56A), or the stressosome kinase RsbT (rsbT-N49A). The rsbS S56A and rsbT N49A mutations abolished the response to low pH. The rsbR1-T209A and rsbR1-T241A mutants displayed constitutive σB activity. Mild acid shock upregulates invasion genes inlAB and stimulates epithelial cell invasion, effects that were abolished in mutants with an inactive or overactive stressosome. Overall, the results show that the stressosome is required for acid-induced activation of σB in L. monocytogenes. Furthermore, they show that RsbR1 can function independently of its paralogues and signal transduction requires RsbT-mediated phosphorylation of RsbS on S56 and RsbR1 on T209 but not T175. These insights shed light on the mechanisms of signal transduction that activate the GSR in L. monocytogenes in response to acidic environments, and highlight the role this sensory process in the early stages of the infectious cycle.
Assuntos
Listeria monocytogenes , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Listeria monocytogenes/metabolismo , Fosforilação , Fator sigma/genética , Fator sigma/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Salmonella enterica causes intracellular infections that can be limited to the intestine or spread to deeper tissues. In most cases, intracellular bacteria show moderate growth. How these bacteria face host defenses that recognize peptidoglycan, is poorly understood. Here, we report a high-resolution structural analysis of the minute amounts of peptidoglycan purified from S. enterica serovar Typhimurium (S. Typhimurium) infecting fibroblasts, a cell type in which this pathogen undergoes moderate growth and persists for days intracellularly. The peptidoglycan of these non-proliferating bacteria contains atypical crosslinked muropeptides with stem peptides trimmed at the L-alanine-D-glutamic acid-(γ) or D-glutamic acid-(γ)-meso-diaminopimelic acid motifs, both sensed by intracellular immune receptors. This peptidoglycan has a reduced glycan chain average length and ~30% increase in the L,D-crosslink, a type of bridge shared by all the atypical crosslinked muropeptides identified. The L,D-transpeptidases LdtD (YcbB) and LdtE (YnhG) are responsible for the formation of these L,D-bridges in the peptidoglycan of intracellular bacteria. We also identified in a fraction of muropeptides an unprecedented modification in the peptidoglycan of intracellular S. Typhimurium consisting of the amino alcohol alaninol replacing the terminal (fourth) D-alanine. Alaninol was still detectable in the peptidoglycan of a double mutant lacking LdtD and LdtE, thereby ruling out the contribution of these enzymes to this chemical modification. Remarkably, all multiple mutants tested lacking candidate enzymes that either trim stem peptides or form the L,D-bridges retain the capacity to modify the terminal D-alanine to alaninol and all attenuate NF-κB nuclear translocation. These data inferred a potential role of alaninol-containing muropeptides in attenuating pro-inflammatory signaling, which was confirmed with a synthetic tetrapeptide bearing such amino alcohol. We suggest that the modification of D-alanine to alaninol in the peptidoglycan of non-proliferating intracellular S. Typhimurium is an editing process exploited by this pathogen to evade immune recognition inside host cells.
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Peptidoglicano/química , Peptidoglicano/imunologia , Infecções por Salmonella/imunologia , Salmonella enterica/imunologia , Salmonella enterica/metabolismo , Linhagem Celular , Parede Celular/química , Parede Celular/imunologia , Parede Celular/metabolismo , Humanos , Tolerância Imunológica/imunologia , Peptidoglicano/metabolismoRESUMO
Listeria monocytogenes is a saprophytic gram-positive bacterium, and an opportunistic foodborne pathogen that can produce listeriosis in humans and animals. It has evolved an exceptional ability to adapt to stress conditions encountered in different environments, resulting in a ubiquitous distribution. Because some food preservation methods and disinfection protocols in food-processing environments cannot efficiently prevent contaminations, L. monocytogenes constitutes a threat to human health and a challenge to food safety. In the host, Listeria colonizes the gastrointestinal tract, crosses the intestinal barrier, and disseminates through the blood to target organs. In immunocompromised individuals, the elderly, and pregnant women, the pathogen can cross the blood-brain and placental barriers, leading to neurolisteriosis and materno-fetal listeriosis. Molecular and cell biology studies of infection have proven L. monocytogenes to be a versatile pathogen that deploys unique strategies to invade different cell types, survive and move inside the eukaryotic host cell, and spread from cell to cell. Here, we present the multifaceted Listeria life cycle from a comprehensive perspective. We discuss genetic features of pathogenic Listeria species, analyze factors involved in food contamination, and review bacterial strategies to tolerate stresses encountered both during food processing and along the host's gastrointestinal tract. Then we dissect host-pathogen interactions underlying listerial pathogenesis in mammals from a cell biology and systemic point of view. Finally, we summarize the epidemiology, pathophysiology, and clinical features of listeriosis in humans and animals. This work aims to gather information from different fields crucial for a comprehensive understanding of the pathogenesis of L. monocytogenes.
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Listeria monocytogenes , Listeriose , Adaptação Fisiológica , Idoso , Animais , Feminino , Humanos , Listeria monocytogenes/genética , Listeriose/microbiologia , Mamíferos , Placenta , Gravidez , Virulência/genéticaRESUMO
Biosynthesis and secretion of a complex extracellular matrix (EM) is a hallmark of Salmonella biofilm formation, impacting on its relationship with both the environment and the host. Cellulose is a major component of Salmonella EM. It is considered an anti-virulence factor because it interferes with Salmonella proliferation inside macrophages and virulence in mice. Its synthesis is stimulated by CsgD, the master regulator of biofilm formation in enterobacteria, which in turn is under the control of MlrA, a MerR-like transcription factor. In this work, we identified a SPI-2-encoded Salmonella-specific transcription factor homolog to MlrA, MlrB, that represses transcription of its downstream gene, orf319, and of csgD inside host cells. MlrB is induced in laboratory media mimicking intracellular conditions and inside macrophages, and it is required for intramacrophage proliferation. An increased csgD expression is observed in the absence of MlrB inside host cells. Interestingly, inactivation of the CsgD-controlled cellulose synthase-coding gene restored intramacrophage proliferation to rates comparable to wild-type bacteria in the absence of MlrB. These data indicate that MlrB represses CsgD expression inside host cells and suggest that this repression lowers the activation of the cellulose synthase. Our findings provide a novel link between biofilm formation and Salmonella virulence.
Assuntos
Proteínas de Bactérias/metabolismo , Celulose/metabolismo , Matriz Extracelular/metabolismo , Proteínas de Membrana/metabolismo , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Transativadores/metabolismo , Animais , Proteínas de Bactérias/genética , Biofilmes , Regulação Bacteriana da Expressão Gênica , Interações entre Hospedeiro e Microrganismos , Macrófagos/microbiologia , Proteínas de Membrana/genética , Camundongos , Células RAW 264.7 , Infecções por Salmonella/microbiologia , Salmonella typhimurium/patogenicidade , Transativadores/genética , Transcrição Gênica , Virulência , Fatores de Virulência/metabolismoRESUMO
Listeria monocytogenes is a ubiquitous environmental bacterium and intracellular pathogen that responds to stress using predominantly the alternative sigma factor SigB. Stress is sensed by a multiprotein complex, the stressosome, extensively studied in bacteria grown in nutrient media. Following signal perception, the stressosome triggers a phosphorylation cascade that releases SigB from its anti-sigma factor. Whether the stressosome is activated during the intracellular infection is unknown. Here, we analyzed the subcellular distribution of stressosome proteins in L. monocytogenes located inside epithelial cells following their immunodetection in membrane and cytosolic fractions prepared from intracellular bacteria. Unlike bacteria in laboratory media, intracellular bacteria have a large proportion of the core stressosome protein RsbR1 associated with the membrane. However, another core protein, RsbS, is undetectable. Despite the absence of RsbS, a SigB-dependent reporter revealed that SigB activity increases gradually from early (1 h) to late (6 h) postinfection times. We also found that RsbR1 paralogues attenuate the intensity of the SigB response and that the miniprotein Prli42, reported to tether the stressosome to the membrane in response to oxidative stress, plays no role in associating RsbR1 to the membrane of intracellular bacteria. Altogether, these data indicate that, once inside host cells, the L. monocytogenes stressosome may adopt a unique configuration to sense stress and to activate SigB in the intracellular eukaryotic niche. IMPORTANCE The response to stress mediated by the alternative sigma factor SigB has been extensively characterized in Bacillus subtilis and Listeria monocytogenes. These bacteria sense stress using a supramacromolecular complex, the stressosome, which triggers a cascade that releases SigB from its anti-sigma factor. Despite the fact that many structural data on the complex are available and analyses have been performed in mutants lacking components of the stressosome or the signaling cascade, the integration of the stress signal and the dynamics of stressosome proteins following environmental changes remain poorly understood. Our study provides data at the protein level on essential stressosome components and SigB activity when L. monocytogenes, normally a saprophytic bacterium, adapts to an intracellular lifestyle. Our results support activation of the stressosome complex in intracellular bacteria. The apparent loss of the stressosome core protein RsbS in intracellular L. monocytogenes also challenges current models, favoring the idea of a unique stressosome architecture responding to intracellular host cues.
Assuntos
Proteínas de Bactérias/metabolismo , Células Epiteliais/microbiologia , Listeria monocytogenes/metabolismo , Fator sigma/metabolismo , Estresse Fisiológico , Linhagem Celular , Proliferação de Células , Células Eucarióticas , HumanosRESUMO
Horizontal gene transfer has shaped the evolution of Salmonella enterica as pathogen. Some functions acquired by this mechanism include enzymes involved in peptidoglycan (PG) synthesis and remodeling. Here, we report a novel serovar Typhimurium protein that is absent in non-pathogenic bacteria and bears a LprI functional domain, first reported in a Mycobacterium tuberculosis lipoprotein conferring lysozyme resistance. Based on the presence of such domain, we hypothesized a role of this S. Typhimurium protein in PG metabolism. This protein, which we named ScwA for Salmonella cell wall-related regulator-A, controls positively the levels of the murein lytic transglycosylase MltD. In addition, the levels of other enzymes that cleave bonds in the PG lattice were affected in a mutant lacking ScwA, including a soluble lytic tranglycosylase (Slt), the amidase AmiC, and a few endo- and carboxypeptidases (NlpC, PBP4, and AmpH). The scwA gene has lower G+C content than the genomic average (43.1 vs. 52.2%), supporting acquisition by horizontal transfer. ScwA is located in the periplasm, stabilized by two disulfide bridges, produced preferentially in stationary phase and down-regulated following entry of the pathogen into eukaryotic cells. ScwA deficiency, however, results in a hypervirulent phenotype in the murine typhoid model. Based on these findings, we conclude that ScwA may be exploited by S. Typhimurium to ensure cell envelope homeostasis along the infection and to prevent host overt damage. This role could be accomplished by controlling the production or stability of a reduced number of peptidoglycan hydrolases whose activities result in the release of PG fragments.
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RcsB is a transcriptional regulator that controls expression of numerous genes in enteric bacteria. RcsB accomplishes this role alone or in combination with auxiliary transcriptional factors independently or dependently of phosphorylation. To understand the mechanisms by which RcsB regulates such large number of genes, we performed structural studies as well as in vitro and in vivo functional studies with different RcsB variants. Our structural data reveal that RcsB binds promoters of target genes such as rprA and flhDC in a dimeric active conformation. In this state, the RcsB homodimer docks the DNA-binding domains into the major groove of the DNA, facilitating an initial weak read-out of the target sequence. Interestingly, comparative structural analyses also show that DNA binding may stabilize an active conformation in unphosphorylated RcsB. Furthermore, RNAseq performed in strains expressing wild-type or several RcsB variants provided new insights into the contribution of phosphorylation to gene regulation and assign a potential role of RcsB in controlling iron metabolism. Finally, we delimited the RcsB box for homodimeric active binding to DNA as the sequence TN(G/A)GAN4TC(T/C)NA. This RcsB box was found in promoter, intergenic and intragenic regions, facilitating both increased or decreased gene transcription.
Assuntos
Proteínas de Bactérias/química , Regiões Promotoras Genéticas , Regulon , Salmonella typhimurium/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Modelos Moleculares , Mutação , Fosforilação , Conformação Proteica , Salmonella typhimurium/metabolismo , Transcrição GênicaRESUMO
Listeria monocytogenes responds to environmental stress using a supra-macromolecular complex, the stressosome, to activate the stress sigma factor SigB. The stressosome structure, inferred from in vitro-assembled complexes, consists of the core proteins RsbR (here renamed RsbR1) and RsbS and, the kinase RsbT. The active complex is proposed to be tethered to the membrane and to support RsbR1/RsbS phosphorylation by RsbT and the subsequent release of RsbT following signal perception. Here, we show in actively-growing cells that L. monocytogenes RsbR1 and RsbS localize mostly in the cytosol in a fully phosphorylated state regardless of osmotic stress. RsbT however distributes between cytosolic and membrane-associated pools. The kinase activity of RsbT on RsbR1/RsbS and its requirement for maximal SigB activation in response to osmotic stress were demonstrated in vivo. Cytosolic RsbR1 interacts with RsbT, while this interaction diminishes at the membrane when RsbR1 paralogues (RsbR2, RsbR3 and RsbL) are present. Altogether, the data support a model in which phosphorylated RsbR1/RsbS may sustain basal SigB activity in unstressed cells, probably assuring a rapid increase in such activity in response to stress. Our findings also suggest that in vivo the active RsbR1-RsbS-RsbT complex forms only transiently and that membrane-associated RsbR1 paralogues could modulate its assembly.
Assuntos
Listeria monocytogenes/genética , Pressão Osmótica/fisiologia , Fator sigma/metabolismo , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Listeria monocytogenes/metabolismo , Fosfoproteínas/metabolismo , Fosforilação/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/fisiologia , Estresse Fisiológico/genéticaRESUMO
BACKGROUND: Salmonella causes intracellular infections in humans. Besides quinolones, third generation cephalosporins are first line drugs used for salmonellosis therapy. An unresolved anomaly of this practice involves high relapse rates associated to quinolone- or cephalosporin-susceptible Salmonella isolates in patients that are discharged clinically following initial recovery. Reduced drug accessibility to intracellular locations has been hypothesized to impair pathogen eradication although supporting evidence is lacking in vivo. Here, we uncover a novel penicillin-binding protein as the first Salmonella factor likely contributing to relapse following beta-lactam, mainly ceftriaxone, therapy. METHODS: We used Salmonella enterica serovar Typhimurium mutants lacking the alternative penicillin-binding proteins PBP2SAL or PBP3SAL. Affinity of PBP2SAL and PBP3SAL for beta-lactam antibiotics was tested. Relapse after ceftriaxone therapy was analysed in the murine typhoid model. FINDINGS: S. Typhimurium does not express PBP2SAL or PBP3SAL in the Mueller-Hinton medium used for susceptibility testing. The pathogen produces these PBPs in response to acidic pH and nutrient limitation, conditions found in phagosomes of mammalian cells. PBP3SAL has low affinity for beta-lactams, even at acidic pH. In vitro susceptibility to ceftriaxone at low pH is strongly reduced. S. Typhimurium lacking PBP3SAL was unable to cause relapse in mice following ceftriaxone therapy. INTERPRETATION: The reduced capacity of ceftriaxone to clear S. Typhimurium in vivo is favoured by a switch in beta-lactam targets. This switch, involving production of the less-susceptible PBP3SAL, remains invisible for standard procedures used in clinical therapy. We conclude that eradication of salmonellosis will be possible only upon targeting of PBP3SAL with novel drugs.
Assuntos
Antibacterianos/farmacologia , Ceftriaxona/farmacologia , Farmacorresistência Bacteriana/genética , Regulação Bacteriana da Expressão Gênica , Proteínas de Ligação às Penicilinas/genética , Infecções por Salmonella/tratamento farmacológico , Salmonella typhimurium/genética , Animais , Deleção de Genes , Concentração de Íons de Hidrogênio , Fígado/microbiologia , Fígado/patologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Ligação às Penicilinas/deficiência , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Recidiva , Infecções por Salmonella/microbiologia , Infecções por Salmonella/patologia , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/metabolismo , Salmonella typhimurium/patogenicidade , Baço/microbiologia , Baço/patologiaRESUMO
This Article contains a URL for a publically available whole-genome browser ( http://nterm.listeriomics.pasteur.fr ). However, due to technical constraint, this website has been replaced with an alternative ( https://listeriomics.pasteur.fr ).
RESUMO
Following colonization of host tissues, bacterial pathogens encounter new niches in which they must gain access to nutrients and cope with stresses and defence signals generated by the host. For some pathogens, the adaptation to a new 'within-host' lifestyle involves modifications of envelope components that bear molecular patterns normally recognized by the host innate immune system. These new modified patterns limit host recognition, therefore promoting immune evasion and pathogenicity. In this review, we describe how envelope components like the peptidoglycan or lipopolysaccharide can be altered within the host to impair responses triggered by pattern recognition receptors (PRR). We also discuss the few cases reported to date of chemical modifications that occur in the envelope of some intracellular bacterial pathogens when they reside inside eukaryotic cells. These envelope alterations may have evolved due to the sentinel role performed by PRRs over pathogen-specific molecular patterns. The available data indicate that only selected pathogens seem to evade recognition due to 'within-host' envelope changes, with most of them displaying such patterns also in non host environments. Given the importance of these alterations, future studies should focus in the responsible pathogen regulators, most yet unknown, that could be targeted to prevent immune evasion.
Assuntos
Cápsulas Bacterianas/química , Lipopolissacarídeos/imunologia , Antígeno 96 de Linfócito/imunologia , Proteínas NLR/imunologia , Peptidoglicano/imunologia , Receptores Toll-Like/imunologia , Animais , Bactérias/crescimento & desenvolvimento , Bactérias/imunologia , Cápsulas Bacterianas/imunologia , Células Eucarióticas/imunologia , Células Eucarióticas/microbiologia , Regulação da Expressão Gênica , Humanos , Evasão da Resposta Imune , Imunidade Inata , Inflamassomos/imunologia , Inflamassomos/metabolismo , Lipopolissacarídeos/metabolismo , Antígeno 96 de Linfócito/genética , Proteínas NLR/genética , Peptidoglicano/metabolismo , Transdução de Sinais , Receptores Toll-Like/genéticaRESUMO
Bacterial cell division has been studied extensively under laboratory conditions. Despite being a key event in the bacterial cell cycle, cell division has not been explored in vivo in bacterial pathogens interacting with their hosts. We discovered in Salmonella enterica serovar Typhimurium a gene absent in nonpathogenic bacteria and encoding a peptidoglycan synthase with 63% identity to penicillin-binding protein 3 (PBP3). PBP3 is an essential cell division-specific peptidoglycan synthase that builds the septum required to separate daughter cells. Since S Typhimurium carries genes that encode a PBP3 paralog-which we named PBP3SAL-and PBP3, we hypothesized that there are different cell division events in host and nonhost environments. To test this, we generated S Typhimurium isogenic mutants lacking PBP3SAL or the hitherto considered essential PBP3. While PBP3 alone promotes cell division under all conditions tested, the mutant producing only PBP3SAL proliferates under acidic conditions (pH ≤ 5.8) but does not divide at neutral pH. PBP3SAL production is tightly regulated with increased levels as bacteria grow in media acidified up to pH 4.0 and in intracellular bacteria infecting eukaryotic cells. PBP3SAL activity is also strictly dependent on acidic pH, as shown by beta-lactam antibiotic binding assays. Live-cell imaging microscopy revealed that PBP3SAL alone is sufficient for S Typhimurium to divide within phagosomes of the eukaryotic cell. Additionally, we detected much larger amounts of PBP3SAL than those of PBP3 in vivo in bacteria colonizing mouse target organs. Therefore, PBP3SAL evolved in S Typhimurium as a specialized peptidoglycan synthase promoting cell division in the acidic intraphagosomal environment.IMPORTANCE During bacterial cell division, daughter cells separate by a transversal structure known as the division septum. The septum is a continuum of the cell wall and therefore is composed of membrane(s) and a peptidoglycan layer. To date, actively growing bacteria were reported to have only a "cell division-specific" peptidoglycan synthase required for the last steps of septum formation and consequently, essential for bacterial life. Here, we discovered that Salmonella enterica has two peptidoglycan synthases capable of synthesizing the division septum. One of these enzymes, PBP3SAL, is present only in bacterial pathogens and evolved in Salmonella to function exclusively in acidic environments. PBP3SAL is used preferentially by Salmonella to promote cell division in vivo in mouse target organs and inside acidified phagosomes. Our data challenge the concept of only one essential cell division-specific peptidoglycan synthase and demonstrate that pathogens can divide in defined host locations using alternative mechanisms.
Assuntos
Divisão Celular , Parede Celular/metabolismo , Proteínas de Ligação às Penicilinas/metabolismo , Fagossomos/microbiologia , Salmonella typhimurium/crescimento & desenvolvimento , Estruturas Animais/microbiologia , Animais , Linhagem Celular , Meios de Cultura/química , Deleção de Genes , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Proteínas de Ligação às Penicilinas/genética , Salmonelose Animal/microbiologia , Salmonella typhimurium/genéticaRESUMO
More than a century ago, infections by Salmonella were already associated with foodborne enteric diseases with high morbidity in humans and cattle. Intestinal inflammation and diarrhea are hallmarks of infections caused by nontyphoidal Salmonella serovars, and these pathologies facilitate pathogen transmission to the environment. In those early times, physicians and microbiologists also realized that typhoid and paratyphoid fever caused by some Salmonella serovars could be transmitted by "carriers," individuals outwardly healthy or at most suffering from some minor chronic complaint. In his pioneering study of the nontyphoidal serovar Typhimurium in 1967, Takeuchi published the first images of intracellular bacteria enclosed by membrane-bound vacuoles in the initial stages of the intestinal epithelium penetration. These compartments, called Salmonella-containing vacuoles, are highly dynamic phagosomes with differing biogenesis depending on the host cell type. Single-cell studies involving real-time imaging and gene expression profiling, together with new approaches based on genetic reporters sensitive to growth rate, have uncovered unprecedented heterogeneous responses in intracellular bacteria. Subpopulations of intracellular bacteria displaying fast, reduced, or no growth, as well as cytosolic and intravacuolar bacteria, have been reported in both in vitro and in vivo infection models. Recent investigations, most of them focused on the serovar Typhimurium, point to the selection of persisting bacteria inside macrophages or following an autophagy attack in fibroblasts. Here, we discuss these heterogeneous intracellular lifestyles and speculate on how these disparate behaviors may impact host-to-host transmissibility of Salmonella serovars.
Assuntos
Infecções por Salmonella/microbiologia , Infecções por Salmonella/transmissão , Salmonella/fisiologia , Animais , Autofagia , Interações Hospedeiro-Patógeno , Humanos , Salmonella/genética , Infecções por Salmonella/fisiopatologiaRESUMO
To adapt to changing environments, bacteria have evolved numerous pathways that activate stress response genes. In Gram-positive bacteria, the stressosome, a cytoplasmic complex, relays external cues and activates the sigma B regulon. The stressosome is structurally well-characterized in Bacillus, but how it senses stress remains elusive. Here, we report a genome-wide N-terminomic approach in Listeria that strikingly led to the discovery of 19 internal translation initiation sites and 6 miniproteins, among which one, Prli42, is conserved in Firmicutes. Prli42 is membrane-anchored and interacts with orthologues of Bacillus stressosome components. We reconstituted the Listeria stressosome in vitro and visualized its supramolecular structure by electron microscopy. Analysis of a series of Prli42 mutants demonstrated that Prli42 is important for sigma B activation, bacterial growth following oxidative stress and for survival in macrophages. Taken together, our N-terminonic approach unveiled Prli42 as a long-sought link between stress and the stressosome.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Firmicutes/genética , Regulação Bacteriana da Expressão Gênica , Listeria monocytogenes/genética , Estresse Fisiológico/genética , Firmicutes/metabolismo , Genoma Bacteriano , Listeria monocytogenes/metabolismo , Membranas/química , Membranas/metabolismo , Processamento de Proteína Pós-Traducional/genética , Proteômica/métodos , Regulon/genética , Fator sigma/genética , Transdução de SinaisRESUMO
High-throughput RNA sequencing (RNA-Seq) has uncovered hundreds of small RNAs and complex modes of RNA regulation in every bacterium analyzed to date. This complexity agrees with the adaptability of most bacteria to varied environments including, in the case of pathogens, the new niches encountered in the host. Recent RNA-Seq studies have analyzed simultaneously gene expression in the intracellular pathogen Salmonella enterica and infected host cells at population and single-cell level. Distinct polarization states or interferon responses in the infected macrophage were linked to variable growth rates or activities of defined virulence regulators in intra-phagosomal bacteria. Intracellular Salmonella, however, exhibit disparate intracellular lifestyles depending the host cell, ranging from a hyper-replicative cytosolic state in epithelial cells to a non-replicative intra-phagosomal condition in varied host cell types. The basis of such diverse pathogen-host communications could be examined by RNA-Seq studies in single intracellular Salmonella cells, certainly a challenge for future investigations.
Assuntos
RNA Bacteriano/genética , Salmonella enterica/fisiologia , Análise de Sequência de RNA/métodos , Animais , Células Epiteliais/microbiologia , Regulação Bacteriana da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Interações Hospedeiro-Patógeno , Humanos , Macrófagos/microbiologia , Salmonella enterica/genéticaRESUMO
Bacterial pathogenicity deeply depends on the ability to secrete virulence factors that bind specific targets on host cells and manipulate host responses. The Gram-positive bacterium Listeria monocytogenes is a human foodborne pathogen that remains a serious public health concern. To transport proteins across its cell envelope, this facultative intracellular pathogen engages a set of specialized secretion systems. Here we show that L. monocytogenes EGDe uses a specialized secretion system, named ESX-1, to secrete EsxA, a homolog of the virulence determinants ESAT-6 and EsxA of Mycobacterium tuberculosis and Staphylococcus aureus, respectively. Our data show that the L. monocytogenes ESX-1 secretion system and its substrates are dispensable for bacterial invasion and intracellular multiplication in eukaryotic cell lines. Surprisingly, we found that the EssC-dependent secretion of EsxA has a detrimental effect on L. monocytogenes in vivo infection.
Assuntos
Proteínas de Bactérias/genética , Sistemas de Secreção Bacterianos/genética , Listeria monocytogenes/genética , Listeria monocytogenes/patogenicidade , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos/metabolismo , Linhagem Celular , Humanos , Listeria monocytogenes/crescimento & desenvolvimento , Listeria monocytogenes/metabolismo , Mycobacterium tuberculosis/patogenicidade , Staphylococcus aureus/patogenicidade , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
IgaA is an integral inner membrane protein that was discovered as repressor of the RcsCDB phosphorelay system in the intracellular pathogen Salmonella enterica serovar Typhimurium. The RcsCDB system, conserved in many members of the family Enterobacteriaceae, regulates expression of varied processes including motility, biofilm formation, virulence and response to envelope stress. IgaA is an essential protein to which, in response to envelope perturbation, the outer membrane lipoprotein RcsF has been proposed to bind in order to activate the RcsCDB phosphorelay. Envelope stress has also been reported to be sensed by a surface exposed domain of RcsF. These observations support a tight control of the RcsCDB system by RcsF and IgaA via mechanisms that, however, remain unknown. Interestingly, RcsF and IgaA have four conserved cysteine residues in loops exposed to the periplasmic space. Two non-consecutive disulfide bonds were shown to be required for RcsF function. Here, we report mutagenesis studies supporting the presence of one disulfide bond (C404-C425) in the major periplasmic loop of IgaA that is essential for repression of the RcsCDB phosphorelay. Our data therefore suggest that the redox state of the periplasm may be critical for the control of the RcsCDB system by its two upstream regulators, RcsF and IgaA.
RESUMO
After crossing the intestinal barrier, the bacterial pathogen Listeria monocytogenes disseminates via the blood to the liver, spleen, brain and placenta. Transcriptomic studies have shown that L. monocytogenes changes expression of many genes during this blood-stage. However, no comparable data at the protein level are known. As main interactors with the environment, we focused in surface proteins produced by L. monocytogenes in an ex vivo bovine blood model. Bacteria exposed to blood alter selectively the amount of several surface proteins compared with bacteria grown in laboratory media. Increased levels were detected for Lmo0514 and Internalin A, two surface proteins covalently bound to peptidoglycan, and the moonlighting protein alcohol-acetaldehyde dehydrogenase, also known as Lap for 'Listeria adhesion protein'. Lmo0514, induced by L. monocytogenes inside epithelial cells, is required for survival in plasma and for virulence in mice at early infection stages. Lmo0514 is also important to cope with low pH stress. By contrast, L. monocytogenes down-regulates other surface proteins following exposure to blood and plasma such as Internalin I. These data provide evidence for remodelling of the L. monocytogenes cell surface during the blood-stage, which it could facilitate pathogen dissemination to deep organs.