1 Department of Food Science, Cornell University, Ithaca, NY 14853, USA
2 Department of Microbiology and Immunology, Cornell University, Ithaca, NY 14853, USA
Correspondence
Kathryn J. Boor
kjb4{at}cornell.edu
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ABSTRACT |
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INTRODUCTION |
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Recently, the alternative sigma factor, B, which was initially identified as responsible for general stress responses in Gram-positive bacteria (Hecker & Volker, 2001
), has also been associated with invasion capabilities in L. monocytogenes. Specifically, a
B-dependent promoter has been identified upstream of inlA (P4inlA) (Kazmierczak et al., 2003
). Loss of
B resulted in reduced inlA expression and InlA levels in stationary-phase cells (Kim et al., 2004
). However, the presence of a putative
B-dependent promoter upstream of inlB (P2inlB) (Kazmierczak et al., 2003
) suggests that contributions of
B to L. monocytogenes invasion may not be solely limited to modulation of inlA expression.
To further study the role of B in L. monocytogenes invasion, we analysed invasion capabilities of various mutant strains bearing combinations of in-frame deletions in inlA, inlB and sigB in the human enterocyte Caco-2 and hepatocyte HepG-2 cell lines. Previous identification of the
B-dependent P2prfA promoter (Nadon et al., 2002
) suggested that the contributions of
B to L. monocytogenes virulence gene expression might be at least partially mediated through PrfA. To quantify the relative functional contributions of
B and PrfA, invasion capabilities of
sigB and
prfA strains were compared. We also measured
B-mediated contributions to expression of multiple genes reported to contribute to L. monocytogenes invasion and virulence using TaqMan quantitative reverse transcriptase polymerase chain reactions (qRT-PCR). Specifically, relative expression of inlA, inlB, prfA, iap, act A and clpC was measured in both the wild-type and
sigB backgrounds. Here, we present evidence that
B is a major contributor to L. monocytogenes invasion, primarily through modulation of expression of inlA and inlB.
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METHODS |
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Cell culture and invasion assay.
The human colorectal epithelial cell line Caco-2 (ATCC HTB-37) and human hepatic epithelial cell line HepG-2 (ATCC HB-8065) were cultivated at 37 °C in a cell culture incubator at 8095 % relative humidity under 5 % CO2. Caco-2 cells were cultured in EMEM (Eagle's Minimum Essential Medium with Earle's Salts) supplemented with 20 % fetal bovine serum (FBS), 1 % non-essential amino acids, 1 % sodium pyruvate, and antibiotics (penicillin G 100 units ml1; streptomycin 100 µg ml1). HepG-2 cells were cultured in DMEM (Dulbecco's Modified Eagle's Medium) supplemented with 10 % FBS, 1 % non-essential amino acids, 1 % sodium pyruvate, and antibiotics (penicillin G 100 units ml1, streptomycin 100 µg ml1). Two days prior to infection, 1·5x105 Caco-2 and 7·5x105 HepG-2 cells in media without antibiotics were seeded into each of six (35 mm diameter) tissue culture plate wells that contained three 12 mm glass coverslips. Host cells were grown to confluence for 2 days. Thirty minutes before infection, the medium in each well was replaced with pre-warmed fresh medium without antibiotics. For infection, approximately 108 c.f.u. of exponential- or stationary-phase bacteria were inoculated onto the host cell monolayer in each well. Host cells were washed with PBS at 30 min post-infection and prewarmed fresh medium containing 50 µg gentamicin sulfate ml1 was added. The number of internalized bacteria per coverslip was determined at 1 h post-infection by lysing infected cells in distilled water and plating appropriate serial dilutions of lysates onto LB (LuriaBertani) agar plates.
Total RNA preparation and TaqMan qRT-PCR.
Total RNA was purified from exponential- and stationary-phase bacterial cells using the RNAprotect/RNeasy Midi kit (Qiagen) and treated with RNase-free DNase as described by Sue et al. (2004). qRT-PCR was performed as described previously (Sue et al., 2004
) using the ABI Prism 7000 Sequence Detection System (Applied Biosystems). TaqMan primers and probes were designed using Primer Express software (Applied Biosystems) according to the manufacturer's guidelines. The primers and probes for rpoB and inlA were reported previously (Sue et al., 2004
); those created for this study are listed in Table 2
. All primers were tested in PCRs with 10403S genomic DNA as template and the amplification products were evaluated by gel electrophoresis. For each RNA sample, the control transcript (rpoB or gap mRNA) and target gene transcripts (prfA, clpC, inlA, inlB, actA or iap mRNAs) were transcribed in the same 96-well plate, and the resulting cDNAs were quantified by real-time PCR. Specifically, RT-PCR reactions were performed using the TaqMan One-Step RT-PCR Master Mix Reagents kit according to the manufacturer's instructions (Applied Biosystems) using 25 ng total RNA with the following reaction conditions: 1 cycle at 48 °C for 30 min, 1 cycle at 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. Transcript levels for each gene (i.e. cDNA copy numbers) were determined as the difference between the experimental reactions and the corresponding reverse-transcriptase-negative controls, which were used to quantify the amount of contaminating L. monocytogenes DNA in each reaction. Standard curves for each gene were generated by using serial dilutions of 10403S genomic DNA template that had been prepared as described by Flamm et al. (1984)
. Absolute cDNA copy numbers, which were calculated based on genomic DNA standard curves to reflect mRNA levels for each gene present in each RNA sample, were used for subsequent analyses.
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RESULTS AND DISCUSSION |
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The inlB strain was more defective in invasion of HepG-2 cells than of Caco-2 cells, as previously reported (Dramsi et al., 1995
) (Table 3
); however, the
inlB invasion defect was less severe than that of
inlA in both cell lines (Table 3
). Further, in contrast to the
inlA strain, the relative invasion defect associated with the
inlB strain was similar regardless of growth phase (Table 3
). Specifically, invasion of the
inlB strain was reduced 2- and 2-fold in Caco-2 cells and 4- and 5-fold in HepG-2 cells with exponential- and stationary-phase bacteria, respectively. In the absence of both inlA and inlB, L. monocytogenes invasion was reduced 38- and 43-fold in Caco-2 cells, and 9- and 125-fold in HepG-2 cells with exponential- and stationary-phase bacteria, respectively (Table 3
). While loss of both InlA and InlB greatly reduced L. monocytogenes invasion of Caco-2 cells independently of growth phase, the effects of their loss on HepG-2 invasion were more pronounced with stationary-phase bacteria.
InlB is required for L. monocytogenes entry into hepatocytes (Dramsi et al., 2004). As previously reported (Dramsi et al., 1995
), we also found that the L. monocytogenes 10403S
inlB strain was more defective in invasion of HepG-2 cells than that of Caco-2 cells. However, our
inlB strain was less defective at invasion than the
inlA strain in both HepG-2 and Caco-2 cells (Table 3
). This observation contrasts with the results of Dramsi et al. (1995)
, who showed a threefold reduced invasion capacity for a L. monocytogenes EGD
inlB strain relative to that of an EGD
inlA strain in HepG-2 cells. The most likely explanations for this discrepancy are: (i) as InlA-mediated invasion is affected by bacterial growth phase (Table 3
), differences in bacterial growth and harvest conditions between the experiments are likely to affect relative strain invasion capacity; and (ii) the relative roles of the internalin proteins in mediating host cell entry may differ between L. monocytogenes EGD and 10403S.
Invasion by the sigB strain was significantly reduced compared with that of the wild-type strain (Table 3
). Loss of
B resulted in a greater bacterial invasion defect with HepG-2 cells than with Caco-2 cells. In Caco-2 cells, the ability of the
sigB strain to invade was reduced 3- and 4-fold with exponential- and stationary-phase bacteria, respectively. In HepG-2 cells, invasion of the
sigB strain was decreased 6- and 59-fold with exponential- and stationary-phase bacteria, respectively, which is essentially equivalent to the invasion defect resulting from the
inlA mutation in this host cell line.
Loss of B in the
inlA background resulted in a further reduction of exponential-phase bacterial invasion in Caco-2 cells (Table 3
), while loss of
B in the
inlB background resulted in a further reduction in L. monocytogenes invasion in both host cell lines, regardless of bacterial growth phase (Table 3
). Loss of
B in the
inlAB background did not contribute to a further reduction in L. monocytogenes invasion (Table 3
). Taken together, these results suggest that
B contributes to invasion of L. monocytogenes into Caco-2 and HepG-2 cells predominantly by directly affecting InlA- and InlB-mediated invasion pathways rather than through indirect mechanisms, such as those that might be mediated by PrfA. Further, the invasion defects resulting from additional loss of
B are essentially equivalent to those resulting from loss of InlA in the
inlB background for L. monocytogenes invasion into HepG-2 cells.
Invasion capabilities of the prfA strain were reduced relative to those of the wild-type strain in both Caco-2 and HepG-2 cells (3- and 2-fold decrease in Caco-2 cells, and 8- and 2-fold decrease in HepG-2 cells with exponential- and stationary-phase bacteria, respectively; Table 3
). The ability of the
prfA strain to invade these host cells was similar to that of the
sigB strain for exponential-phase bacteria, but greater than that of the
sigB strain for stationary-phase bacteria (Table 3
). These results suggest that the contribution of PrfA to L. monocytogenes invasion differs with growth phase, with a greater relative contribution in exponential-phase than in stationary-phase bacteria.
B modulates expression of inlA and inlB
To determine the effect of B on the expression of genes responsible for L. monocytogenes entry into non-phagocytic cells, we analysed relative expression of six selected genes (prfA, clpC, inlA, inlB, actA and iap) in the wild-type and
sigB strains using TaqMan qRT-PCR. To provide two independent assessments of relative gene expression patterns, mRNA collected from two different housekeeping genes, rpoB and gap, was used to normalize target gene expression data. Although expression patterns generated by normalizing target gene transcripts with those of each housekeeping gene were similar, they were not identical (Fig. 1
). To provide the most conservative interpretation of the data, transcript levels representing a target gene under a given condition were only deemed different from those of the gene under a different condition (e.g. exponential- vs stationary-phase) or in a different background (wild-type vs
sigB) if levels were statistically significantly different by both normalizing analyses.
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qRT-PCR analyses showed that in exponential-phase L. monocytogenes, levels of inlA expression were similarly low in the wild-type and the sigB strains (Fig. 1
). In stationary phase, however, inlA expression was significantly up-regulated in the wild-type strain (919-fold) (P<0·05), but remained at a level similar to that in exponential phase in the
sigB strain (Fig. 1
). These results show that
B plays a critical role for stationary-phase up-regulation of inlA. As with inlA (Fig. 1
), exponential-phase inlB expression was similarly low in the wild-type and the
sigB strains (Fig. 1
), and stationary-phase inlB expression was significantly up-regulated in the wild-type (36-fold) (P<0·05), but not in the
sigB strain (Fig. 1
). These findings demonstrate that
B contributes to inlB expression as well as to inlA expression in stationary-phase bacteria.
Relative expression of prfA was evaluated for the wild-type and sigB strains, as the P2prfA promoter has been shown to be
B-dependent (Nadon et al., 2002
) and as regulation of inlA and inlB also is influenced by a PrfA-dependent mechanism (Dramsi et al., 1993
; Lingnau et al., 1995
). Although prfA expression appeared higher in the wild-type strain than in the
sigB strain, both in exponential and in stationary phase (Fig. 1
), the differences were not statistically significant. Further, prfA expression also was not statistically different in exponential- and stationary-phase cells. These results suggest that the
B-regulated P2prfA promoter does not play a predominant role in prfA expression under the conditions examined in this study, and that increased transcriptional activation of prfA is not required for increased expression of inlA and inlB in stationary-phase L. monocytogenes cells. These data provide additional support for the conclusion that
B contributes to L. monocytogenes invasion primarily by directly affecting inlA and inlB expression, rather than through indirect effects mediated by PrfA.
B does not make major contributions to clpC, actA or iap expression under the conditions examined in this study
To quantify contributions of B to expression of multiple L. monocytogenes invasion genes, iap, actA and clpC transcripts were measured using qRT-PCR in both wild-type and
sigB backgrounds. iap encodes a major surface protein, p60, which is indirectly involved in invasion (Wuenscher et al., 1993
) and actA also participates in L. monocytogenes invasion (Alvarez-Dominguez et al., 1997
). clpC reportedly contributes to L. monocytogenes virulence (Rouquette et al., 1996
, 1998
) and influences expression of inlA, inlB and actA (Nair et al., 2000a
). Regulation of clpC appears to be very complex, involving several regulators, including CtsR, PrfA and
B (Nair et al., 2000b
; Ripio et al., 1998
).
Expression of clpC and iap was significantly affected by bacterial growth phase (Fig. 1). Specifically, transcripts for both genes were present at significantly higher levels in stationary-phase bacteria than in exponential-phase bacteria for both the wild-type and
sigB strains (P<0·05). Although clpC and iap transcripts appeared to be present at higher levels in the wild-type strain than in the
sigB strain (Fig. 1a
), the differences were not statistically significant at the 95 % confidence level when expression data were normalized by gap (Fig. 1b
), suggesting that
B is not a predominant contributor to clpC or iap expression under the conditions examined in this study. In contrast, while actA also appeared to be affected by bacterial growth phase, actA transcripts were present at higher levels in exponential-phase than in stationary-phase bacteria for both strains when data were normalized by rpoB (Fig. 1
). Transcript levels were not lower in the
sigB strain, and did not differ significantly between the wild-type and
sigB strains in data normalized by gap, suggesting that
B is not a positive regulator of actA expression in L. monocytogenes. These results suggest that any contributions of Iap, ActA and ClpC to L. monocytogenes invasion are predominantly independent of
B, providing further support for the hypothesis that
B-mediated invasion effects occur primarily through its regulation of expression of inlA and inlB.
Conclusions
The ability of L. monocytogenes to invade non-phagocytic cells allows the organism to breach host barriers, and hence is critical for systemic listeriosis. Our results demonstrate that B significantly contributes to L. monocytogenes invasion of human enterocytes and hepatocytes, predominantly through InlA- and InlB-mediated pathways, as shown by both invasion and TaqMan qRT-PCR assay results. Specifically, we have shown that while stationary-phase expression of inlA and inlB is significantly enhanced (918-fold for inlA expression; 36-fold for inlB expression) in the wild-type strain relative to that in exponential phase (Fig. 1
), stationary-phase expression of inlA and inlB does not increase in the
sigB strain. Further, loss of
B did not significantly reduce expression of Iap, ActA or ClpC, each of which have been associated with L. monocytogenes invasion. Our data support a model in which invasion defects associated with loss of
B result from loss of
B-mediated transcription of the inlAB locus, with relatively minor, if any, indirect effects resulting from
B-dependent expression of prfA. In support of this hypothesis, we have demonstrated dramatically reduced expression of both inlA and inlB in stationary-phase
sigB cells despite essentially wild-type expression levels for prfA (Fig. 1
). However, our results do not rule out the possibility that the relative role of PrfA in invasion reflects growth-phase-dependent changes in PrfA activity to a greater extent than it reflects changes in prfA transcriptional activation, as PrfA is known to exist in both active and inactive forms (Renzoni et al., 1997
). While additional studies will be necessary to fully attribute the relative contributions of PrfA- and
B-mediated mechanisms to invasion, the results presented in this study clearly highlight critical contributions of
B to L. monocytogenes invasion into non-phagocytic cells.
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ACKNOWLEDGEMENTS |
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Received 26 March 2005;
revised 4 July 2005;
accepted 19 July 2005.
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