RESUMO
Respiration-deficient Staphylococcus aureus small-colony variants (SCVs) frequently cause persistent infections, which necessitates they acquire iron, yet how SCVs obtain iron remains unknown. To address this, we created a stable hemB mutant from S. aureus USA300 strain LAC. The hemB SCV utilized exogenously supplied hemin but was attenuated for growth under conditions of iron starvation. Transcriptome sequencing (RNA-seq) showed that both wild-type (WT) S. aureus and the hemB mutant sense and respond to iron starvation; however, growth assays show that the hemB mutant is defective for siderophore-mediated iron acquisition. Indeed, the hemB SCV demonstrated limited utilization of endogenous staphyloferrin B or exogenously provided staphyloferrin A, deferoxamine mesylate (Desferal), and epinephrine. Direct measurement of intracellular ATP in hemB and WT S. aureus revealed that both strains can generate comparable levels of ATP during exponential growth, suggesting defects in ATP production cannot account for the inability to efficiently utilize siderophores. Defective siderophore utilization by hemB bacteria was also evident in vivo, as administration of Desferal failed to promote hemB bacterial growth in every organ analyzed except for the kidneys. In support of the hypothesis that S. aureus accesses heme in kidney abscesses, in vitro analyses revealed that increased hemin availability enables hemB bacteria to utilize siderophores for growth when iron availability is restricted. Taken together, our data support the conclusion that hemin is used not only as an iron source itself but also as a nutrient that promotes utilization of siderophore-iron complexes. IMPORTANCE S. aureus small-colony variants (SCVs) are associated with chronic recurrent infection and worsened clinical outcome. SCVs persist within the host despite administration of antibiotics. This study yields insight into how S. aureus SCVs acquire iron, which during infection of a host is a difficult-to-acquire metal nutrient. Under hemin-limited conditions, hemB S. aureus is impaired for siderophore-dependent growth, and in agreement, murine infection indicates that hemin-deficient SCVs meet their nutritional requirement for iron through utilization of hemin. Importantly, we demonstrate that hemB SCVs rely upon hemin as a nutrient to promote siderophore utilization. Therefore, perturbation of heme biosynthesis and/or utilization represents a viable to strategy to mitigate the ability of SCV bacteria to acquire siderophore-bound iron during infection.
Assuntos
Proteínas de Bactérias/metabolismo , Heme/metabolismo , Ferro/administração & dosagem , Sideróforos/metabolismo , Staphylococcus aureus/metabolismo , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/fisiologia , Variação Genética , Ferro/metabolismoRESUMO
Inoculation studies are important when assessing microbial survival and growth in food products. These studies typically involve the pregrowth of multiple strains of a target pathogen under a single condition; this emphasizes strain diversity. To gain a better understanding of the impacts of strain diversity ("nature") and pregrowth conditions ("nurture") on subsequent bacterial growth in foods, we assessed the growth and survival of Salmonella enterica (n = 5), Escherichia coli (n = 6), and Listeria (n = 5) inoculated onto tomatoes, precut lettuce, and cantaloupe rind, respectively. Pregrowth conditions included (i) 37°C to stationary phase (baseline), (ii) low pH, (iii) high salt, (iv) reduced water activity, (v) log phase, (vi) minimal medium, and (vii) 21°C. Inoculated tomatoes were incubated at 21°C; lettuce and cantaloupe were incubated at 7°C. Bacterial counts were assessed over three phases, including initial reduction (phase 1), change in bacterial numbers over the first 24 h of incubation (phase 2), and change over the 7-day incubation (phase 3). E. coli showed overall decline in counts (<1 log) over the 7-day period, except for a <1-log increase after pregrowth in high salt and to mid-log phase. In contrast, S. enterica and Listeria showed regrowth after an initial reduction. Pregrowth conditions had a substantial and significant effect on all three phases of S. enterica and E. coli population dynamics on inoculated produce, whereas strain did not show a significant effect. For Listeria, both pregrowth conditions and strain affected changes in phase 2 but not phases 1 and 3.IMPORTANCE Our findings suggest that inclusion of multiple pregrowth conditions in inoculation studies can best capture the range of growth and survival patterns expected for Salmonella enterica and Escherichia coli present on produce. This is particularly important for fresh and fresh-cut produce, where stress conditions encountered by pathogens prior to contamination can vary widely, making selection of a typical pregrowth condition virtually impossible. Pathogen growth and survival data generated using multiple pregrowth conditions will allow for more robust microbial risk assessments that account more accurately for uncertainty.
Assuntos
Cucumis melo/microbiologia , Escherichia coli/crescimento & desenvolvimento , Lactuca/microbiologia , Listeria/crescimento & desenvolvimento , Salmonella enterica/crescimento & desenvolvimento , Solanum lycopersicum/microbiologia , Contagem de Colônia Microbiana , Microbiologia de AlimentosRESUMO
The ability of bacteria to adapt to stress depends on the conditional expression of specific sets of genes. Bacillus subtilis encodes seven extracytoplasmic function (ECF) sigma (σ) factors that regulate functions important for survival under conditions eliciting cell envelope stress. Of these, four have been studied in detail: σM, σW, σX and σV. These four σ factors recognize overlapping sets of promoters, although the sequences that determine this overlapping recognition are incompletely understood. A major role in promoter selectivity has been ascribed to the core -10 and -35 promoter elements. Here, we demonstrate that a homopolymeric T-tract motif, proximal to the -35 element, functions in combination with the core promoter sequences to determine selectivity for ECF sigma factors. This motif is most critical for promoter activation by σV, and contributes variably to activation by σM, σX and σW. We propose that this motif, which is a feature of the deduced promoter consensus for a subset of ECF σ factors from many species, imparts intrinsic DNA curvature to influence promoter activity. The differential effect of this region among ECF σ factors thereby provides a mechanism to modulate the nature and extent of regulon overlap.
Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/genética , Perfilação da Expressão Gênica/métodos , Regiões Promotoras Genéticas/genética , Fator sigma/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Sequência de Bases , Parede Celular/metabolismo , Regulação Bacteriana da Expressão Gênica , Mutação , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Regulon/genética , Fator sigma/metabolismoRESUMO
BACKGROUND: Alternative σ factors are important transcriptional regulators in bacteria. While σ(B) has been shown to control a large regulon and play important roles in stress response and virulence in the pathogen Listeria monocytogenes, the function of σ(H) has not yet been well defined in Listeria, even though σ(H) controls a large regulon in the closely related non-pathogenic Bacillus subtilis. RESULTS: Using RNA-seq characterization of a L. monocytogenes strain with deletions of all 4 genes encoding alternative σ factors (ΔBCHL), which was further modified to overexpress sigH (ΔBCHL::P rha -sigH), we identified 6 transcription units (TUs) that are transcribed from σ(H)-dependent promoters. Five of these TUs had not been previously identified. Identification of these promoters was facilitated by use of a bio-informatics approach that compared normalized RNA-seq coverage (NRC), between ΔBCHL::P rha -sigH and a ΔBCHL control, using sliding windows of 51 nt along the whole genome rather than comparing NRC calculated only for whole genes. Interestingly, we found that three operons that encode competence genes (comGABCDEFG, comEABC, coiA) are transcribed from σ(H)-dependent promoters. While these promoters were highly conserved in L. monocytogenes, none of them were found in all Listeria spp. and coiA and its σ(H)-dependent promoter were only found in L. monocytogenes. CONCLUSIONS: Our data indicate that a number of L. monocytogenes competence genes are regulated by σ(H). This σ(H)-dependent regulation of competence related genes is conserved in the pathogen L. monocytogenes, but not in other non-pathogenic Listeria strains. Combined with prior data that indicated a role of σ(H) in virulence in a mouse model, this suggests a possible novel role of σ(H)-dependent competence genes in L. monocytogenes virulence. Development and implementation of a sliding window approach to identify differential transcription using RNA-seq data, not only allowed for identification of σ(H)-dependent promoters, but also provides a general approach for sensitive identification of differentially transcribed promoters and genes, particularly for genes that are transcribed from multiple promoter elements only some of which show differential transcription.
Assuntos
Biologia Computacional/métodos , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Listeria monocytogenes/genética , RNA Bacteriano , Análise de Sequência de RNA , Fator sigma/genética , Sequência de Bases , Sequência Conservada , Motivos de Nucleotídeos , Óperon , Matrizes de Pontuação de Posição Específica , Regiões Promotoras Genéticas , Alinhamento de Sequência , Fator sigma/metabolismoRESUMO
Gram-positive bacteria are ubiquitous and diverse microorganisms that can survive and sometimes even thrive in continuously changing environments. The key to such resilience is the ability of members of a population to respond and adjust to dynamic conditions in the environment. In bacteria, such responses and adjustments are mediated, at least in part, through appropriate changes in the bacterial transcriptome in response to the conditions encountered. Resilience is important for bacterial survival in diverse, complex, and rapidly changing environments and requires coordinated networks that integrate individual, mechanistic responses to environmental cues to enable overall metabolic homeostasis. In many Gram-positive bacteria, a key transcriptional regulator of the response to changing environmental conditions is the alternative sigma factor σ(B) σ(B) has been characterized in a subset of Gram-positive bacteria, including the genera Bacillus, Listeria, and Staphylococcus Recent insight from next-generation-sequencing results indicates that σ(B)-dependent regulation of gene expression contributes to resilience, i.e., the coordination of complex networks responsive to environmental changes. This review explores contributions of σ(B) to resilience in Bacillus, Listeria, and Staphylococcus and illustrates recently described regulatory functions of σ(B).
Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Bactérias Gram-Positivas/metabolismo , Fator sigma/metabolismo , Proteínas de Bactérias/genética , Bactérias Gram-Positivas/genética , Fator sigma/genéticaRESUMO
The sigma-like factor YvrI and coregulator YvrHa activate transcription from a small set of conserved promoters in Bacillus subtilis. We report here that these two proteins independently contribute sigma-region 2 and sigma-region 4 functions to a holoenzyme-promoter DNA complex. YvrI binds RNA polymerase (RNAP) through a region 4 interaction with the beta-subunit flap domain and mediates specific promoter recognition but cannot initiate DNA melting at the -10 promoter element. Conversely, YvrHa possesses sequence similarity to a conserved core-binding motif in sigma-region 2 and binds to the N-terminal coiled-coil element in the RNAP beta'-subunit previously implicated in interaction with region 2 of sigma-factors. YvrHa plays an essential role in stabilizing the open complex and interacts specifically with the N-terminus of YvrI. Based on these results, we propose that YvrHa is situated in the transcription complex proximal to the -10 element of the promoter, whereas YvrI is responsible for -35 region recognition. This system presents an unusual example of a two-subunit bacterial sigma-factor.
Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Fator sigma/genética , Ativação Transcricional , DNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Regiões Promotoras Genéticas , Subunidades Proteicas , Fator sigma/fisiologiaRESUMO
Cold-smoked salmon is a ready-to-eat food product capable of supporting Listeria monocytogenes growth at refrigeration temperatures. While the FDA-approved antimicrobial nisin can be used to mitigate L. monocytogenes contamination, stresses associated with cold-smoked salmon and the associated processing environments may reduce nisin efficacy. A previous study in our laboratory showed that, at high inoculation levels, pre-exposure of L. monocytogenes to sublethal concentrations of quaternary ammonium compounds had an overall detrimental effect on nisin efficacy. The objective of this study was to investigate the impact of nisin concentration and storage temperature on nisin efficacy against L. monocytogenes inoculated on salmon at natural contamination levels. Three L. monocytogenes strains were pre-grown in the presence of sublethal levels of benzalkonium chloride prior to inoculation at ~102 CFU/g on salmon slices that were pre-treated with either 0, 25, or 250 ppm nisin, followed by vacuum-packing and incubation at 4 or 7°C for up to 30 days. L. monocytogenes was enumerated on days 1, 15, and 30 using direct plating and/or most probable number methods. A hurdle model was constructed to describe the odds of complete elimination of L. monocytogenes on salmon and the level of L. monocytogenes when complete elimination was not achieved. Our data showed that (i) nisin efficacy (defined as L. monocytogenes reduction relative to the untreated control) was concentration-dependent with increased efficacy at 250 ppm nisin, and that (ii) 250 ppm nisin treatments led to a reduction in L. monocytogenes prevalence, independent of storage temperature and serotype; this effect of nisin could only be identified since low inoculation levels were used. While lower storage temperatures (i.e., 4°C) yielded lowered absolute L. monocytogenes counts on days 15 and 30 (as compared to 7°C), nisin efficacy did not differ between these two temperatures. Finally, the serotype 1/2b strain was found to be more susceptible to nisin compared with serotype 1/2a and 4b strains on samples incubated at 7°C or treated with 25 ppm nisin. This variation of nisin susceptibility across serotypes, which is affected by both the storage temperature and nisin concentration, needs to be considered while evaluating the efficacy of nisin.
RESUMO
ABSTRACT: Although public health risk assessments for Listeria monocytogenes (Lm) have been published for various foods, firm-level decision making on interventions targeting Lm involves considerations of both public health and enterprise risks. Smoked seafood is a ready-to-eat product with a high incidence of Lm contamination and has been associated with several recalls. We used cold-smoked salmon as a model product to develop a decision support tool (the regulatory and recall risk [3R] model) to estimate (i) baseline regulatory and recall (RR) risks (i.e., overall risks of a lot sampled and found positive for Lm, e.g., by food regulatory agencies) due to Lm contamination and (ii) the RR risk reduction that can be achieved through interventions with underlying mechanisms such as reducing the prevalence and/or level of Lm and retarding or preventing Lm growth. Given that a set number of samples (e.g., 10) are tested for a given lot, the RR risk equals the likelihood of detecting Lm in at least one sample. Under the baseline scenario, which assumes a 4% Lm prevalence and no interventions, the median predicted RR risk for a given production lot was 0.333 (95% credible interval: 0.288, 0.384) when 10 25-g samples were tested. Nisin treatments, which reduce both the prevalence and initial level of Lm, reduced RR risks in a concentration-dependent manner to 0.109 (0.074, 0.146) with 5 ppm, 0.049 (0.024, 0.083) with 10 ppm, and 0.017 (0.007, 0.033) with 20 ppm. In general, more effective reduction in RR risks can be achieved by reducing Lm prevalence than by retarding Lm growth; the RR risk was reduced to 0.182 (0.153, 0.213) by a 50% prevalence reduction but to only 0.313 (0.268, 0.367) by bacteriostatic growth inhibitors. Sensitivity analysis indicated that prevalence and initial level of Lm and storage temperature have the greatest impact on predicting RR risks, suggesting that reliable data for these parameters will improve model performance.
Assuntos
Listeria monocytogenes , Animais , Contagem de Colônia Microbiana , Contaminação de Alimentos/análise , Microbiologia de Alimentos , Conservação de Alimentos , Salmão , Alimentos Marinhos/análiseRESUMO
The seven extracytoplasmic function (ECF) sigma (σ) factors of Bacillus subtilis are broadly implicated in resistance to antibiotics and other cell envelope stressors mediated, in part, by regulation of cell envelope synthesis and modification enzymes. We here define the regulon of σ(V) as including at least 20 operons, many of which are also regulated by σ(M), σ(X), or σ(W). The σ(V) regulon is strongly and specifically induced by lysozyme, and this induction is key to the intrinsic resistance of B. subtilis to lysozyme. Strains with null mutations in either sigV or all seven ECF σ factor genes (Δ7ECF) have essentially equal increases in sensitivity to lysozyme. Induction of σ(V) in the Δ7ECF background restores lysozyme resistance, whereas induction of σ(M), σ(X), or σ(W) does not. Lysozyme resistance results from the ability of σ(V) to activate the transcription of two operons: the autoregulated sigV-rsiV-oatA-yrhK operon and dltABCDE. Genetic analyses reveal that oatA and dlt are largely redundant with respect to lysozyme sensitivity: single mutants are not affected in lysozyme sensitivity, whereas an oatA dltA double mutant is as sensitive as a sigV null strain. Moreover, the sigV oatA dltA triple mutant is no more sensitive than the oatA dltA double mutant, indicating that there are no other σ(V)-dependent genes necessary for lysozyme resistance. Thus, we suggest that σ(V) confers lysozyme resistance by the activation of two cell wall modification pathways: O-acetylation of peptidoglycan catalyzed by OatA and D-alanylation of teichoic acids by DltABCDE.
Assuntos
Bacillus subtilis/metabolismo , Parede Celular/metabolismo , Farmacorresistência Bacteriana , Peptidoglicano/metabolismo , Fator sigma/metabolismo , Ácidos Teicoicos/metabolismo , Acetilação , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/genética , Parede Celular/genética , Regulação Bacteriana da Expressão Gênica , Muramidase/metabolismo , Muramidase/farmacologia , Óperon , Fator sigma/genéticaRESUMO
The foodborne pathogen Listeria monocytogenes is able to survive across a wide range of intra- and extra-host environments by appropriately modulating gene expression patterns in response to different stimuli. Positive Regulatory Factor A (PrfA) is the major transcriptional regulator of virulence gene expression in L. monocytogenes. It has long been known that activated charcoal is required to induce the expression of PrfA-regulated genes in complex media, such as Brain Heart Infusion (BHI), but not in chemically defined media. In this study, we show that the expression of the PrfA-regulated hly, which encodes listeriolysin O, is induced 5- and 8-fold in L. monocytogenes cells grown in Chelex-treated BHI (Ch-BHI) and in the presence of activated charcoal (AC-BHI), respectively, relative to cells grown in BHI medium. Specifically, we show that metal ions present in BHI broth plays a role in the reduced expression of the PrfA regulon. In addition, we show that expression of hly is induced when the levels of bioavailable extra- or intercellular iron are reduced. L. monocytogenes cells grown Ch-BHI and AC-BHI media showed similar levels of resistance to the iron-activated antibiotic, streptonigrin, indicating that activated charcoal reduces the intracellular labile iron pool. Metal depletion and exogenously added glutathione contributed synergistically to PrfA-regulated gene expression since glutathione further increased hly expression in metal-depleted BHI but not in BHI medium. Analyses of transcriptional reporter fusion expression patterns revealed that genes in the PrfA regulon are differentially expressed in response to metal depletion, metal excess and exogenous glutathione. Our results suggest that metal ion abundance plays a role in modulating expression of PrfA-regulated virulence genes in L. monocytogenes.
Assuntos
Toxinas Bacterianas/genética , Carvão Vegetal/farmacologia , Proteínas de Choque Térmico/genética , Proteínas Hemolisinas/genética , Listeria monocytogenes/crescimento & desenvolvimento , Fatores de Terminação de Peptídeos/genética , Poliestirenos/farmacologia , Polivinil/farmacologia , Proteínas de Bactérias/genética , Meios de Cultura/química , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Glutationa/metabolismo , Ferro/química , Listeria monocytogenes/efeitos dos fármacos , Listeria monocytogenes/genética , Listeria monocytogenes/patogenicidade , Estreptonigrina/farmacologia , Virulência/efeitos dos fármacos , Zinco/químicaRESUMO
Coagulase-negative staphylococci and Staphylococcus aureus colonize similar niches in mammals and conceivably compete for space and nutrients. Here, we report that a coagulase-negative staphylococcus, Staphylococcus chromogenes ATCC43764, synthesizes and secretes 6-thioguanine (6-TG), a purine analog that suppresses S. aureus growth by inhibiting de novo purine biosynthesis. We identify a 6-TG biosynthetic gene cluster in S. chromogenes and other coagulase-negative staphylococci including S. epidermidis, S. pseudintermedius and S. capitis. Recombinant S. aureus strains harbouring this operon produce 6-TG and, when used in subcutaneous co-infections in mice with virulent S. aureus USA300, protect the host from necrotic lesion formation. Used prophylactically, 6-TG reduces necrotic skin lesions in mice infected with USA300, and this effect is mediated by abrogation of toxin production. RNAseq analyses reveal that 6-TG downregulates expression of genes coding for purine biosynthesis, the accessory gene regulator (agr) and ribosomal proteins in S. aureus, providing an explanation for its effect on toxin production.
Assuntos
Infecções Cutâneas Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus/genética , Staphylococcus/metabolismo , Tioguanina/metabolismo , Animais , Proteínas de Bactérias/biossíntese , Coagulase/deficiência , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Purinas/biossíntese , Proteínas Ribossômicas/biossíntese , Staphylococcus aureus/patogenicidade , Staphylococcus capitis/metabolismo , Staphylococcus epidermidis/metabolismo , Tioguanina/farmacologia , Transativadores/biossínteseRESUMO
The aim of this study was to determine the presence of Helicobacter pylori cytotoxin-associated gene (cagA)/vacuolating cytotoxin gene (vacA) among patients with chronic gastritis in Cuba and Venezuela. Gastric antrum biopsies were taken for culture, DNA extraction and PCR analysis. Amplification of vacA and cagA segments was performed using two regions of cagA: 349 bp were amplified with the F1/B1 primers and the remaining 335 bp were amplified with the B7629/B7628 primers. The VA1-F/VA1-R set of primers was used to amplify the 259-bp (s1) or 286-bp (s2) product and the VAG-R/VAG-F set of primers was used to amplify the 567-bp (m1) or 642-bp (m2) regions of vacA. cagA was detected in 87% of the antral samples from Cuban patients and 80.3% of those from Venezuelan patients. All possible combinations of vacA regions were found, with the exception of s2/m1. The predominant combination found in both countries was s1/m1. The percentage of cagA+ strains was increased by the use of a second set of primers and a greater number of strains was amplified with the B7629/B7628 primers in the Cuban patients (p = 0.0001). There was no significant difference between the presence of the allelic variants of vacA and cagA in both populations. The predominant genotype was cagA+/s1m1 in both countries. The results support the necessary investigation of isolates circulating among the human population in each region.
Assuntos
Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Gastrite/microbiologia , Infecções por Helicobacter/microbiologia , Helicobacter pylori/genética , Adulto , Idoso , Doença Crônica , Cuba , DNA Bacteriano/genética , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Venezuela , Adulto JovemRESUMO
Listeria monocytogenes is a human pathogen that is commonly found in environments associated with cold-smoked salmon. Nisin is a natural antimicrobial that can be used as a food preservative. While nisin is active against a number of Gram-positive bacteria, including L. monocytogenes, environmental stresses encountered in cold-smoked salmon processing facilities might affect L. monocytogenes' nisin susceptibility. The objective of this study was to investigate the effect of seafood-relevant pre-growth conditions and L. monocytogenes strain diversity on nisin treatment efficacy on cold-smoked salmon. Six L. monocytogenes strains representing serotypes most commonly associated with cold-smoked salmon (1/2a, 1/2b, and 4b) were initially pre-grown under a number of seafood-relevant conditions and challenged with nisin in growth media modified to represent the characteristics of cold-smoked salmon. The pre-growth conditions with the lowest mean log reduction due to nisin and the highest strain-to-strain variability were selected for experiments on cold-smoked salmon; these included: (i) 4.65% w.p. NaCl ("NaCl"); (ii) pH = 6.1 ("pH"); (iii) 0.5 µg/ml benzalkonium chloride ("Quat"); and a control ("BHI"). Cold-smoked salmon slices with or without nisin were inoculated with L. monocytogenes pre-grown in one of the conditions above, vacuum-packed, and incubated at 7 °C. L. monocytogenes were enumerated on days 1, 15, and 30. A linear mixed effects model was constructed to investigate the effect of pre-growth condition, day in storage, serotype, source of isolation as well as their interactions on nisin efficacy against L. monocytogenes. Compared to pre-growth in "BHI", significant reduction (P < 0.05) in nisin efficacy was induced by pre-growth in "pH" and "Quat" on both days 15 and 30, and by pre-growth in "NaCl" on day 30, indicating a time-dependent cross-protection effect. Additionally, an effect of L. monocytogenes' serotype on the cross-protection to nisin was observed; pre-growth in "pH" significantly reduced nisin efficacy against serotype 1/2a and 4b strains, but not against 1/2b strains. In conclusion, pre-exposure to mildly acidic environment, high salt content, and sublethal concentrations of quaternary ammonium compounds, is likely to provide cross-protection against a subsequent nisin treatment of L. monocytogenes on cold-smoked salmon. Therefore, challenge studies that use pre-growth in "BHI", as well as more susceptible L. monocytogenes strains, may overestimate the efficacy of nisin as a control strategy for cold-smoked salmon.
Assuntos
Antibacterianos/farmacologia , Conservantes de Alimentos/farmacologia , Listeria monocytogenes/efeitos dos fármacos , Nisina/farmacologia , Salmão/microbiologia , Animais , Contagem de Colônia Microbiana , Conservação de Alimentos/métodos , Humanos , Listeria monocytogenes/crescimento & desenvolvimento , Listeria monocytogenes/isolamento & purificação , Alimentos Marinhos/microbiologiaRESUMO
The foodborne pathogen Listeria monocytogenes can modulate its transcriptome and proteome to ensure its survival during transmission through vastly differing environmental conditions. While L. monocytogenes utilizes a large array of regulators to achieve survival and growth in different intra- and extrahost environments, the alternative sigma factor σB and the transcriptional activator of virulence genes protein PrfA are two key transcriptional regulators essential for responding to environmental stress conditions and for host infection. Importantly, emerging evidence suggests that the shift from extrahost environments to the host gastrointestinal tract and, subsequently, to intracellular environments requires regulatory interplay between σB and PrfA at transcriptional, posttranscriptional, and protein activity levels. Here, we review the current evidence for cross talk and interplay between σB and PrfA and their respective regulons and highlight the plasticity of σB and PrfA cross talk and the role of this cross talk in facilitating successful transition of L. monocytogenes from diverse extrahost to diverse extra- and intracellular host environments.
Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Listeria monocytogenes/genética , Fatores de Terminação de Peptídeos/genética , Fator sigma/genética , Transdução de Sinais , Proteínas de Bactérias/metabolismo , Perfilação da Expressão Gênica , Humanos , Listeria monocytogenes/patogenicidade , Fatores de Terminação de Peptídeos/metabolismo , Fator sigma/metabolismo , Estresse Fisiológico , VirulênciaRESUMO
Effective control of foodborne pathogens on produce requires science-based validation of interventions and control strategies, which typically involves challenge studies with a set of bacterial strains representing the target pathogens or appropriate surrogates. In order to facilitate these types of studies, a produce-relevant strain collection was assembled to represent strains from produce outbreaks or pre-harvest environments, including Listeria monocytogenes (n = 11), Salmonella enterica (n = 23), shiga-toxin producing Escherichia coli (STEC) (n = 13), and possible surrogate organisms (n = 8); all strains were characterized by whole genome sequencing (WGS). Strain diversity was assured by including the 10 most common S. enterica serotypes, L. monocytogenes lineages I-IV, and E. coli O157 as well as selected "non-O157" STEC serotypes. As it has previously been shown that strains and genetic lineages of a pathogen may differ in their ability to survive different stress conditions, a subset of representative strains for each "pathogen group" (e.g., Salmonella, STEC) was selected and assessed for survival of exposure to peroxyacetic acid (PAA) using strains pre-grown under different conditions including (i) low pH, (ii) high salt, (iii) reduced water activity, (iv) different growth phases, (v) minimal medium, and (vi) different temperatures (21°C, 37°C). The results showed that across the three pathogen groups pre-growth conditions had a larger effect on bacterial reduction after PAA exposure as compared to strain diversity. Interestingly, bacteria exposed to salt stress (4.5% NaCl) consistently showed the least reduction after exposure to PAA; however, for STEC, strains pre-grown at 21°C were as tolerant to PAA exposure as strains pre-grown under salt stress. Overall, our data suggests that challenge studies conducted with multi-strain cocktails (pre-grown under a single specific condition) may not necessarily reflect the relevant phenotypic range needed to appropriately assess different intervention strategies.
RESUMO
Aim: Among the alternative sigma factors of Listeria monocytogenes, σB controls the largest regulon. The aim of this study was to perform a comprehensive review of σB-regulated genes, and the functions they confer. Materials & methods: A systematic search of PubMed and Web of Knowledge was carried out to identify members of the σB regulon based on experimental evidence of σB-dependent transcription and presence of a consensus σB-dependent promoter. Results: The literature review identified σB-dependent transcription units encompassing 304 genes encoding different functions including stress response and virulence. Conclusion: Our review supports the well-known roles of σB in virulence and stress response and provides new insight into novel roles for σB in metabolism and overall resilience of L. monocytogenes.
Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Listeria monocytogenes/genética , Metabolismo , Regulon , Fator sigma/metabolismo , Estresse Fisiológico , Listeria monocytogenes/patogenicidade , Listeria monocytogenes/fisiologia , VirulênciaRESUMO
Listeria monocytogenes uses a variety of transcriptional regulation strategies to adapt to the extra-host environment, the gastrointestinal tract, and the intracellular host environment. While the alternative sigma factor SigB has been proposed to be a key transcriptional regulator that facilitates L. monocytogenes adaptation to the gastrointestinal environment, the L. monocytogenes' transcriptional response to bile exposure is not well-understood. RNA-seq characterization of the bile stimulon was performed in two L. monocytogenes strains representing lineages I and II. Exposure to bile at pH 5.5 elicited a large transcriptomic response with ~16 and 23% of genes showing differential transcription in 10403S and H7858, respectively. The bile stimulon includes genes involved in motility and cell wall modification mechanisms, as well as genes in the PrfA regulon, which likely facilitate survival during the gastrointestinal stages of infection that follow bile exposure. The fact that bile exposure induced the PrfA regulon, but did not induce further upregulation of the SigB regulon (beyond that expected by exposure to pH 5.5), suggests a model where at the earlier stages of gastrointestinal infection (e.g., acid exposure in the stomach), SigB-dependent gene expression plays an important role. Subsequent exposure to bile induces the PrfA regulon, potentially priming L. monocytogenes for subsequent intracellular infection stages. Some members of the bile stimulon showed lineage- or strain-specific distribution when 27 Listeria genomes were analyzed. Even though sigB null mutants showed increased sensitivity to bile, the SigB regulon was not found to be upregulated in response to bile beyond levels expected by exposure to pH 5.5. Comparison of wildtype and corresponding ΔsigB strains newly identified 26 SigB-dependent genes, all with upstream putative SigB-dependent promoters.
RESUMO
Among Listeria monocytogenes' four alternative σ factors, σB controls the largest regulon. As σB-dependent transcription of some genes may be masked by overlaps among regulons, and as some σB-dependent genes are expressed only under very specific conditions, we hypothesized that the σB regulon is not yet fully defined. To further extend our understanding of the σB regulon, we used RNA-seq to identify σB-dependent genes in an L. monocytogenes strain that expresses σB following rhamnose induction, and in which genes encoding the other alternative sigma factors have been deleted. Analysis of RNA-seq data with multiple bioinformatics approaches, including a sliding window method that detects differentially transcribed 5' untranslated regions (UTRs), identified 105 σB-dependent transcription units (TUs) comprising 201 genes preceded by σB-dependent promoters. Of these 105 TUs, 7 TUs comprising 15 genes had not been identified previously as σB-dependent. An additional 23 genes not reported previously as σB-dependent were identified in 9 previously recognized σB-dependent TUs. Overall, 38 of these 201 genes had not been identified previously as members of the L. monocytogenes σB regulon. These newly identified σB-dependent genes encode proteins annotated as being involved in transcriptional regulation, oxidative and osmotic stress response, and in metabolism of energy, carbon and nucleotides. In total, 18 putative σB-dependent promoters were newly identified. Interestingly, a number of genes previously identified as σB-dependent did not show significant evidence for σB-dependent transcription in our experiments. Based on promoter analyses, a number of these genes showed evidence for co-regulation by σB and other transcriptional factors, suggesting that some σB-dependent genes require additional transcriptional regulators along with σB for transcription. Over-expression of a single alternative sigma factor in the absence of all other alternative sigma factors allowed us to: (i) identify new σB-dependent functions in L. monocytogenes, such as regulation of genes involved in 1,2-propanediol utilization (LMRG_00594-LMRG_00611) and biosynthesis of pyrimidine nucleotides (LMRG_00978-LMRG_00985); and (ii) identify new σB-dependent genes involved in stress response and pathogenesis functions. These data further support that σB not only regulates stress response functions, but also plays a broad role in L. monocytogenes homeostasis and resilience.
RESUMO
During host infection, the foodborne pathogen Listeria monocytogenes must sense and respond to rapidly changing environmental conditions. Two transcriptional regulators, the alternative sigma factor B (σB) and the Positive Regulatory Factor A (PrfA), are key contributors to the transcriptomic responses that enable bacterial survival in the host gastrointestinal tract and invasion of host duodenal cells. Increases in temperature and osmolarity induce activity of these proteins; such conditions may be encountered in food matrices as well as within the host gastrointestinal tract. Differences in PrfA and σB activity between individual cells might affect the fate of a cell during host invasion, therefore, we hypothesized that PrfA and σB activities differ among individual cells under heat and salt stress. We used fluorescent reporter fusions to determine the relative proportions of cells with active σB or PrfA following exposure to 45°C heat or 4% NaCl. Activities of both PrfA and σB were induced stochastically, with fluorescence levels ranging from below detection to high among individual cells. The proportion of cells with active PrfA was significantly higher than the proportion with active σB under all tested conditions; under some conditions, nearly all cells had active PrfA. Our findings further support the growing body of evidence illustrating the stochastic nature of bacterial gene expression under conditions that are relevant for host invasion via food-borne, oral infection.
RESUMO
Our understanding of how pathogens shape their gene expression profiles in response to environmental changes is ever growing. Advances in Bioinformatics have made it possible to model complex systems and integrate data from variable sources into one large regulatory network. In these analyses, regulatory networks are typically broken down into regulatory motifs such as feed-forward loops (FFL) or auto-regulatory feedbacks, which serves to simplify the structure, while the functional implications of different regulatory motifs allow to make informed assumptions about the function of a specific regulatory pathway. Here we review the basic concepts of network features and use this language to break down the regulatory networks that govern the interactions between the main regulators of stress response, virulence, and transmission in Listeria monocytogenes. We point out the advantage that taking a "systems approach" could have for our understanding of gene functions, the detection of distant regulatory inputs, interspecies comparisons, and co-expression.