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 σB, 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 σB, 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 σB 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.
Get full access to this article
View all access options for this article.
References
1.
AndersenJB, RoldgaardB, ChristensenB, LichtT. Oxygen restriction increases the infective potential of Listeria monocytogenes in vitro in Caco-2 cells and in vivo in guinea pigs. BMC Microbiol, 2007; 7:55.
2.
AutretN, RaynaudC, DubailI, BercheP, CharbitA. Identification of the agr locus of Listeria monocytogenes: role in bacterial virulence. Infect Immun, 2003; 71:4463–4471.
3.
BakardjievAI, StacyBA, FisherSJ, PortnoyDA. Listeriosis in the pregnant guinea pig: a model of vertical transmission. Infect Immun, 2004; 72:489–497.
4.
BeckerLA, EvansSN, HutkinsRW, BensonAK. Role of Sigma B in adaptation of Listeria monocytogenes to growth at low temperature. J Bacteriol, 2000; 182:7083–7087.
5.
BenninkR, PeetersM, Van den MaegdenberghV, GeypensB, RutgeertsP, De RooM, MortelmansL. Evaluation of small-bowel transit for solid and liquid test meal in healthy men and women. Eur J Nucl Med Mol Imaging, 1999; 26:1560–1566.
6.
BishopDK, HinrichsDJ. Adoptive transfer of immunity to Listeria monocytogenes. The influence of in vitro stimulation on lymphocyte subset requirements. J Immunol, 1987; 139:2005–2009.
7.
BrondstedL, KallipolitisBH, IngmerH, KnochelS. kdpE and a putative RsbQ homologue contribute to growth of Listeria monocytogenes at high osmolarity and low temperature. FEMS Microbiol Lett, 2003; 219:233–239.
8.
CamilliA, TilneyLG, PortnoyDA. Dual roles of plcA in Listeria monocytogenes pathogenesis. Mol Microbiol, 1993; 8:143–157.
9.
CetinMS, ZhangC, HutkinsRW, BensonAK. Regulation of transcription of compatible solute transporters by the general stress sigma factor, Sigma B, in Listeria monocytogenes. J Bacteriol, 2004; 186:794–802.
10.
ChanYC, BoorKJ, WiedmannM. Sigma B-dependent and -independent mechanisms contribute to transcription of Listeria monocytogenes cold stress genes during cold shock and cold growth. Appl Environ Microbiol, 2007a. 73:6019–6029.
11.
ChanYC, RaengpradubS, BoorKJ, WiedmannM. Microarray-based characterization of the Listeria monocytogenes cold regulon in log- and stationary-phase cells. Appl Environ Microbiol, 2007b. 73:6484–6498.
12.
ChaturongakulS, BoorKJ. Sigma B activation under environmental and energy stress conditions in Listeria monocytogenes. Appl Environ Microbiol, 2006; 72:5197–5203.
13.
ChengLW, PortnoyDA. Drosophila S2 cells: an alternative infection model for Listeria monocytogenes. Cell Microbiol, 2003; 5:875–885.
14.
CotterPD, EmersonN, GahanCGM, HillC. Identification and disruption of lisRK, a genetic locus encoding a two-component signal transduction system involved in stress tolerance and virulence in Listeria monocytogenes. J Bacteriol, 1999; 181:6840–6843.
15.
Di BonaventuraG, PiccolominiR, PaludiD, D'OrioV, VergaraA, ConterM, IanieriA. Influence of temperature on biofilm formation by Listeria monocytogenes on various food-contact surfaces: relationship with motility and cell surface hydrophobicity. J Appl Microbiol, 2008; 104:1552–1561.
16.
DonsL, ErikssonE, JinY, RottenbergME, KristenssonK, LarsenCN, BrescianiJ, OlsenJE. Role of flagellin and the two-component CheA/CheY system of Listeria monocytogenes in host cell invasion and virulence. Infect Immun, 2004; 72:3237–3244.
17.
DramsiS, KocksC, ForestierC, CossartP. Internalin-mediated invasion of epithelial cells by Listeria monocytogenes is regulated by the bacterial growth state, temperature and the pleiotropic activator PrfA. Mol Microbiol, 1993; 9:931–941.
18.
FreitagNE, RongL, PortnoyDA. Regulation of the PrfA transcriptional activator of Listeria monocytogenes: multiple promoter elements contribute to intracellular growth and cell-to-cell spread. Infect Immun, 1993; 61:2537–2544.
19.
GarnerMR, JamesKE, CallahanMC, WiedmannM, BoorKJ. Exposure to salt and organic acids increases the ability of Listeria monocytogenes to invade Caco-2 cells but decreases its ability to survive gastric stress. Appl Environ Microbiol, 2006a. 72:5384–5395.
20.
GarnerMR, NjaaBL, WiedmannM, BoorKJ. Sigma B contributes to Listeria monocytogenes gastrointestinal infection but not to systemic spread in the guinea pig infection model. Infect Immun, 2006b. 74:876–886.
21.
GilbrethSE, BensonAK, HutkinsRW. Catabolite repression and virulence gene expression in Listeria monocytogenes. Curr Microbiol, 2004; 49:95–98.
22.
GrundlingA, BurrackLS, BouwerHGA, HigginsDE. From the cover: Listeria monocytogenes regulates flagellar motility gene expression through MogR, a transcriptional repressor required for virulence. PNAS, 2004; 101:12318–12323.
23.
HayashiF, SmithKD, OzinskyA, HawnTR, YiEC, GoodlettDR, EngJK, AkiraS, UnderhillDM, AderemA. The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5. Nature, 2001; 410:1099–1103.
24.
IsbergRR, SwainA, FalkowS. Analysis of expression and thermoregulation of the Yersinia pseudotuberculosis inv gene with hybrid proteins. Infect Immun, 1988; 56:2133–2138.
25.
JohanssonJ, MandinP, RenzoniA, ChiaruttiniC, SpringerM, CossartP. An RNA thermosensor controls expression of virulence genes in Listeria monocytogenes. Cell, 2002; 110:551–561.
26.
KallipolitisBH, IngmerH. Listeria monocytogenes response regulators important for stress tolerance and pathogenesis. FEMS Microbiol Lett, 2001; 204:111–115.
27.
KallipolitisBH, IngmerH, GahanCG, HillC, Sogaard-AndersenL. CesRK, a two-component signal transduction system in Listeria monocytogenes, responds to the presence of cell wall-acting antibiotics and affects {beta}-lactam resistance. Antimicrob Agents Chemother, 2003; 47:3421–3429.
28.
KazmierczakMJ, MithoeSC, BoorKJ, WiedmannM. Listeria monocytogenes Sigma B regulates stress response and virulence functions. J Bacteriol, 2003; 185:5722–5734.
29.
KazmierczakMJ, WiedmannM, BoorKJ. Contributions of Listeria monocytogenes Sigma B and PrfA to expression of virulence and stress response genes during extra- and intracellular growth. Microbiology, 2006; 152:1827–1838.
30.
KimH, BoorKJ, MarquisH. Listeria monocytogenes Sigma B contributes to invasion of human intestinal epithelial cells. Infect Immun, 2004; 72:7374–7378.
31.
KimH, MarquisH, BoorKJ. Sigma B contributes to Listeria monocytogenes invasion by controlling expression of inlA and inlB. Microbiology, 2005; 151:3215–3222.
32.
KnudsenGM, OlsenJE, DonsL. Characterization of DegU, a response regulator in Listeria monocytogenes, involved in regulation of motility and contributes to virulence. FEMS Microbiol Lett, 2004; 240:171–179.
LarsenMH, KallipolitisBH, ChristiansenJK, OlsenJE, IngmerH. The response regulator ResD modulates virulence gene expression in response to carbohydrates in Listeria monocytogenes. Mol Microbiol, 2006; 61:1622–1635.
35.
LecuitM, DramsiS, GottardiC, Fedor-ChaikenM, GumbinerB, CossartP. A single amino acid in E-cadherin responsible for host specificity towards the human pathogen Listeria monocytogenes. EMBO J, 1999; 18:3956–3963.
36.
Leimeister-WachterM, DomannE, ChakrabortyT. The expression of virulence genes in Listeria monocytogenes is thermoregulated. J Bacteriol, 1992; 174:947–952.
37.
LingnauA, DomannE, HudelM, BockM, NichterleinT, WehlandJ, ChakrabortyT. Expression of the Listeria monocytogenes EGD inlA and inlB genes, whose products mediate bacterial entry into tissue culture cell lines, by PrfA-dependent and -independent mechanisms. Infect Immun, 1995; 63:3896–3903.
38.
LiuS, GrahamJE, BigelowL, MorsePDII, WilkinsonBJ. Identification of Listeria monocytogenes genes expressed in response to growth at low temperature. Appl Environ Microbiol, 2002; 68:1697–1705.
MauderN, WilliamsT, FritschF, KuhnM, BeierD. Response regulator DegU of Listeria monocytogenes controls temperature-responsive flagellar gene expression in its unphosphorylated state. J Bacteriol, 2008; 190:4777–4781.
41.
MaurelliAT. Temperature regulation of virulence genes in pathogenic bacteria: a general strategy for human pathogens?Microb Pathog, 1989; 7:1–10.
42.
McGannP, IvanekR, WiedmannM, BoorKJ. Temperature-dependent expression of Listeria monocytogenes internalin and internalin-like genes suggests functional diversity of these proteins among the Listeriae. Appl Environ Microbiol, 2007a. 73:2806–2814.
43.
McGannP, RaengpradubS, IvanekR, WiedmannM, BoorKJ. Differential regulation of Listeria monocytogenes internalin and internalin-like genes by Sigma B and PrfA as revealed by subgenomic microarray analyses. Foodborne Pathog Dis, 2008; 5:417–435.
44.
McGannP, WiedmannM, BoorKJ. The alternative sigma factor Sigma B and the virulence gene regulator PrfA both regulate transcription of Listeria monocytogenes internalins. Appl Environ Microbiol, 2007b. 73:2919–2930.
45.
MeadPS, SlutskerL, DietzV, McCaigLF, BreseeJS, ShapiroC, GriffinPM, TauxeRV. Food-related illness and death in the United States. Emerg Infect Dis, 1999; 5:607–625.
46.
MichelE, MengaudJ, GalsworthyS, CossartP. Characterization of a large motility gene cluster containing the cheR, motAB genes of Listeria monocytogenes and evidence that PrfA downregulates motility genes. FEMS Microbiol Lett, 1998; 169:341–347.
47.
MilenbachsAA, BrownDP, MoorsM, YoungmanP. Carbon-source regulation of virulence gene expression in Listeria monocytogenes. Mol Microbiol, 1997; 23:1075–1085.
48.
MilohanicE, GlaserP, CoppeeJ-Y, FrangeulL, VegaY, Vazquez-BolandJA, KunstF, CossartP, BuchrieserC. Transcriptome analysis of Listeria monocytogenes identifies three groups of genes differently regulated by PrfA. Mol Microbiol, 2003; 47:1613–1625.
49.
OllingerJ, WiedmannM, BoorKJ. SigB- and PrfA-dependent transcription of genes previously classified as putative constituents of the Listeria monocytogenes PrfA Regulon. Foodborne Pathog Dis, 2008; 5:281–293.
50.
O'NeilHS, MarquisH. Listeria monocytogenes flagella are used for motility, not as adhesins, to increase host cell invasion. Infect Immun, 2006; 74:6675–6681.
51.
PalA, LabuzaTP, Diez-GonzalezF. Comparison of primary predictive models to study the growth of Listeria monocytogenes at low temperatures in liquid cultures and selection of fastest growing ribotypes in meat and turkey product slurries. Food Microbiol, 2008; 25:460–470.
52.
RaengpradubS, WiedmannM, BoorKJ. Comparative analysis of the Sigma B-dependent stress responses in Listeria monocytogenes and Listeria innocua strains exposed to selected stress conditions. Appl Environ Microbiol, 2008; 74:158–171.
53.
RaffelsbauerD, BubertA, EngelbrechtF, ScheinpflugJ, SimmA, HessJ, KaufmannSH, GoebelW. The gene cluster inlC2DE of Listeria monocytogenes contains additional new internalin genes and is important for virulence in mice. Mol Gen Genet, 1998; 260:144–158.
54.
RipioMT, BrehmK, LaraM, SuarezM, Vazquez-BolandJA. Glucose-1-phosphate utilization by Listeria monocytogenes is PrfA dependent and coordinately expressed with virulence factors. J Bacteriol, 1997; 179:7174–7180.
55.
RipioMT, DomÌnguez-BernalG, SuárezM, BrehmK, BercheP, Vázquez-BolandJA. Transcriptional activation of virulence genes in wild-type strains of Listeria monocytogenes in response to a change in the extracellular medium composition. Res Microbiol, 1996; 147:371–384.
ShenA, HigginsDE. The MogR transcriptional repressor regulates nonhierarchal expression of flagellar motility genes and virulence in Listeria monocytogenes. PLoS Pathog, 2006; 2:0283–0295.
58.
ShunZ, SilverbergA, ChangC, OuyangP. Dunnett's many-to-one test and least square means. J Biopharm Stat, 2003; 13:17–28.
59.
SleatorRD, HillC. A novel role for the LisRK two-component regulatory system in listerial osmotolerance. Clin Microbiol Infect, 2005; 11:599–601.
60.
SmithK, YoungmanP. Use of a new integrational vector to investigate compartment-specific expression of the Bacillus subtilis spollM gene. Biochimie, 1992; 74:705–711.
61.
SueD, BoorKJ, WiedmannM. Sigma B-dependent expression patterns of compatible solute transporter genes opuCA and lmo1421 and the conjugated bile salt hydrolase gene bsh in Listeria monocytogenes. Microbiology, 2003; 149:3247–3256.
62.
SueD, FinkD, WiedmannM, BoorKJ. Sigma B-dependent gene induction and expression in Listeria monocytogenes during osmotic and acid stress conditions simulating the intestinal environment. Microbiology, 2004; 150:3843–3855.
63.
Toledo-AranaA, DussurgetO, NikitasG, SestoN, Guet-RevilletH, BalestrinoD, LohE, GripenlandJ, TiensuuT, VaitkeviciusK, BarthelemyM, VergassolaM, NahoriM-A, SoubigouG, RegnaultB, CoppeeJ-Y, LecuitM, JohanssonJ, CossartP. The Listeria transcriptional landscape from saprophytism to virulence. Nature, 2009; 459:950–956.
64.
TorresD, BarrierM, BihlF, QuesniauxVJF, MailletI, AkiraS, RyffelB, ErardF. Toll-like Receptor 2 is required for optimal control of Listeria monocytogenes infection. Infect Immun, 2004; 72:2131–2139.
65.
van der VeenS, HainT, WoutersJA, HossainH, de VosWM, AbeeT, ChakrabortyT, Wells-BennikMHJ. The heat-shock response of Listeria monocytogenes comprises genes involved in heat shock, cell division, cell wall synthesis, and the SOS response. Microbiology, 2007; 153:3593–3607.
66.
WaySS, ThompsonLJ, LopesJE, HajjarAM, KollmannTR, FreitagNE, WilsonCB. Characterization of flagellin expression and its role in Listeria monocytogenes infection and immunity. Cell Microbiol, 2004; 6:235–242.
67.
WiedmannM, ArvikTJ, HurleyRJ, BoorKJ. General stress transcription factor Sigma B and its role in acid tolerance and virulence of Listeria monocytogenes. J Bacteriol, 1998; 180:3650–3656.
68.
WilliamsT, BauerS, BeierD, KuhnM. Construction and characterization of Listeria monocytogenes mutants with in-frame deletions in the response regulator genes identified in the genome sequence. Infect Immun, 2005a. 73:3152–3159.
69.
WilliamsT, JosephB, BeierD, GoebelW, KuhnM. Response regulator DegU of Listeria monocytogenes regulates the expression of flagella-specific genes. FEMS Microbiol Lett, 2005b. 252:287–298.
70.
YoungGM, BadgerJL, MillerVL. Motility is required to initiate host cell invasion by Yersinia enterocolitica. Infect Immun, 2000; 68:4323–4326.
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
