Growth temperature-dependent contributions of response regulators, σB, PrfA, and motility factors to Listeria monocytogenes invasion of Caco-2 cells.

Foodborne pathogens encounter rapidly changing environmental conditions during transmission, including exposure to temperatures below 37°C. The goal of this study was to develop a better understanding of the effects of growth temperatures and temperature shifts on regulation of invasion phenotypes and invasion-associated genes in Listeria monocytogenes. We specifically characterized the effects of L. monocytogenes growth at different temperatures (30°C vs. 37°C) on (i) the contributions to Caco-2 invasion of different regulators (including σ(B), PrfA, and 14 response regulators [RRs]) and invasion proteins (i.e., InlA and FlaA), and on (ii) gadA, plcA, inlA, and flaA transcript levels and their regulation. Overall, Caco-2 invasion efficiency was higher for L. monocytogenes grown at 30°C than for bacteria grown at 37°C (p = 0.0051 for the effect of temperature on invasion efficiency; analysis of variance); the increased invasion efficiency of the parent strain 10403S (serotype 1/2a) observed after growth at 30°C persisted for 2.5 h exposure to 37°C. For L. monocytogenes grown at 30°C, the motility RRs DegU and CheY and σ(B), but not PrfA, significantly contributed to Caco-2 invasion efficiency. For L. monocytogenes grown at 37°C, none of the 14 RRs tested significantly contributed to Caco-2 invasion, whereas σ(B) and PrfA contributed synergistically to invasion efficiency. At both growth temperatures there was significant synergism between the contributions to invasion of FlaA and InlA; this synergism was more pronounced after growth at 30°C than at 37°C. Our data show that growth temperature affects invasion efficiency and regulation of virulence-associated genes in L. monocytogenes. These data support increasing evidence that a number of environmental conditions can modulate virulence-associated phenotypes of foodborne bacterial pathogens, including L. monocytogenes.

Physiology and genetics of Listeria monocytogenes survival and growth at cold temperatures.

Listeria monocytogenes is a foodborne pathogen that can cause serious invasive human illness in susceptible patients, notably immunocompromised, pregnant women, and adults > 65 years old. Most human listeriosis cases appear to be caused by consumption of refrigerated ready-to-eat foods that are contaminated with high levels of L. monocytogenes. While initial L. monocytogenes levels in contaminated foods are usually low, the ability of L. monocytogenes to survive and multiply at low temperatures allows it to reach levels high enough to cause human disease, particularly if contaminated foods that allow for L. monocytogenes growth are stored for prolonged times under refrigeration. In this review, relevant knowledge on the physiology and genetics of L. monocytogenes' ability to adapt to and multiply at low temperature will be summarized and discussed, including selected relevant findings on the physiology and genetics of cold adaptation in other Gram-positive bacteria. Further improvement in our understanding of the physiology and genetics of L. monocytogenes cold growth will hopefully enhance our ability to design successful intervention strategies for this foodborne pathogen.

Contributions of two-component regulatory systems, alternative sigma factors, and negative regulators to Listeria monocytogenes cold adaptation and cold growth.

The ability of Listeria monocytogenes to grow at refrigeration temperatures is critical for transmission of this foodborne pathogen. We evaluated the contributions of different transcriptional regulators and two-component regulatory systems to L. monocytogenes cold adaptation and cold growth. L. monocytogenes parent strain 10403S and selected isogenic null mutants in genes encoding four alternative sigma factors (sigB, sigH, sigC, and sigL), two regulators of sigmaB (rsbT and rsbV), two negative regulators (ctsR and hrcA), and 15 two-component response regulators were grown in brain heart infusion broth at 4 degrees C with (i) a high-concentration starting inoculum (10(8) CFU/ml), (ii) a low-concentration starting inoculum (102 CFU/ml), and (iii) a high-concentration starting inoculum of cold-adapted cells. With a starting inoculum of 10(8) CFU/ml, null mutants in genes encoding selected alternative sigma factors (DeltasigH, DeltasigC, and DeltasigL), a negative regulator (DeltactsR), regulators of sigmaB (DeltarsbT and DeltarsbV), and selected two-component response regulators (DeltalisR, Deltalmo1172, and Deltalmo1060) had significantly reduced growth (P < 0.05) compared with the parent strain after 12 days at 4 degrees C. The growth defect for DeltasigL was limited and was not confirmed by optical density (OD600) measurement data. With a starting inoculum of 102 CFU/ml and after monitoring growth at 4 degrees C over 84 days, only the DeltactsR strain had a consistent but limited growth defect; the other mutant strains had either no growth defects or limited growth defects apparent at only one or two of the nine sampling points evaluated during the 84-day growth period (DeltasigB, DeltasigC, and Deltalmo1172). With a 10(8) CFU/ml starting inoculum of cold-adapted cells, none of the mutant strains that had a growth defect when inoculation was performed with cells pregrown at 37 degrees C had reduced growth as compared with the parent strain after 12 days at 4 degrees C, suggesting a specific defect in the ability of these mutant strains to adapt to 4 degrees C after growth at 37 degrees C. Our data indicate (i) selected sigma factors and two-component regulators may contribute to cold adaptation even though two-component regulatory systems, alternative sigma factors, and the negative regulators CtsR and HrcA appear to have limited contributions to L. monocytogenes growth at 4 degrees C in rich media, and (ii) inoculum concentration and pregrowth conditions affect the L. monocytogenes cold-growth phenotype.

SigmaB-dependent and sigmaB-independent mechanisms contribute to transcription of Listeria monocytogenes cold stress genes during cold shock and cold growth.

The role of the stress response regulator sigma(B) (encoded by sigB) in directing the expression of selected putative and confirmed cold response genes was evaluated using Listeria monocytogenes 10403S and an isogenic DeltasigB mutant, which were either cold shocked at 4 degrees C in brain heart infusion (BHI) broth for up to 30 min or grown at 4 degrees C in BHI for 12 days. Transcript levels of the housekeeping genes rpoB and gap, the sigma(B)-dependent genes opuCA and bsh, and the cold stress genes ltrC, oppA, and fri were measured using quantitative reverse transcriptase PCR. Transcriptional start sites for ltrC, oppA, and fri were determined using rapid amplification of cDNA ends PCR. Centrifugation was found to rapidly induce sigma(B)-dependent transcription, which necessitated the use of centrifugation-independent protocols to evaluate the contributions of sigma(B) to transcription during cold shock. Our data confirmed that transcription of the cold stress genes ltrC and fri is at least partially sigma(B) dependent and experimentally identified a sigma(B)-dependent ltrC promoter. In addition, our data indicate that (i) while sigma(B) activity is induced during 30 min of cold shock, this cold shock does not induce the transcription of sigma(B)-dependent or -independent cold shock genes; (ii) sigma(B) is not required for L. monocytogenes growth at 4 degrees C in BHI; and (iii) transcription of the putative cold stress genes opuCA, fri, and oppA is sigma(B) independent during growth at 4 degrees C, while both bsh and ltrC show growth phase and sigma(B)-dependent transcription during growth at 4 degrees C. We conclude that sigma(B)-dependent and sigma(B)-independent mechanisms contribute to the ability of L. monocytogenes to survive and grow at low temperatures.

Microarray-based characterization of the Listeria monocytogenes cold regulon in log- and stationary-phase cells.

Whole-genome microarray experiments were performed to define the Listeria monocytogenes cold growth regulon and to identify genes differentially expressed during growth at 4 and 37 degrees C. Microarray analysis using a stringent cutoff (adjusted P < 0.001; >/=2.0-fold change) revealed 105 and 170 genes that showed higher transcript levels in logarithmic- and stationary-phase cells, respectively, at 4 degrees C than in cells grown at 37 degrees C. A total of 74 and 102 genes showed lower transcript levels in logarithmic- and stationary-phase cells, respectively, grown at 4 degrees C. Genes with higher transcript levels at 4 degrees C in both stationary- and log-phase cells included genes encoding a two-component response regulator (lmo0287), a cold shock protein (cspL), and two RNA helicases (lmo0866 and lmo1722), whereas a number of genes encoding virulence factors and heat shock proteins showed lower transcript levels at 4 degrees C. Selected genes that showed higher transcript levels at 4 degrees C during both stationary and log phases were confirmed by quantitative reverse transcriptase PCR. Our data show that (i) a large number of L. monocytogenes genes are differentially expressed at 4 and 37 degrees C, with more genes showing higher transcript levels than lower transcript levels at 4 degrees C, (ii) L. monocytogenes genes with higher transcript levels at 4 degrees C include a number of genes and operons with previously reported or plausible roles in cold adaptation, and (iii) L. monocytogenes genes with lower transcript levels at 4 degrees C include a number of virulence and virulence-associated genes as well as some heat shock genes.

Comparative analysis of Shigella boydii 18 foodborne outbreak isolate and related enteric bacteria: role of rpoS and adiA in acid stress response.

Shigella boydii CDPH (Chicago Department of Public Health) serotype 18 was implicated in an outbreak of foodborne illness in 1998. The suspected food vehicles were parsley and cilantro imported from Mexico used to prepare bean salad. Previous studies revealed that S. boydii CDPH serotype 18 can survive in bean salad, which contains organic acids and whose pH decreases over time. Acid challenge assays in acidified tryptic soy broth at pH 4.5, acidified Luria-Bertani broth at pH 4.5, and acidified M9 minimal salts medium at pH 2.5 containing amino acids, arginine, or glutamic acid were performed using S. boydii CDPH, S. boydii ATCC 35966, S. flexneri 3136, Escherichia coli O157:H7 dd8872, and E. coli O157:H7 dd642 to compare differences in acid tolerance. Differences in survival of exponential-phase cells were detected in acidified tryptic soy broth and Luria-Bertani broth at pH 4.5. In acidified minimal medium containing arginine, S. boydii strains were able to survive at pH 2.5. The arginine decarboxylase gene (adiA) present in S. boydii is involved in survival at extremely low pH. The discovery of adiA expression in S. boydii serotype 18 by use of an acidified minimal medium challenge and arginine decarboxylase biochemical assay is significant because arginine decarboxylase activity was thought to be unique to E. coli. Sequencing of the rpoS gene from the S. boydii outbreak strain indicates that it is 99% conserved compared with the E. coli K-12 rpoS gene and plays a vital role in survival under acidic conditions.