Table 1.
Bacterial strains and plasmids used in this study.
Table 2.
Primers used in this study.
Figure 1.
Transcriptional overview of chromosomal loci of lmo0515 (A), lmo1580 (B), and lmo2673 (C) derived from sequencing data of L. monocytogenes (data not shown).
σB boxes are depicted as orange arrows, the primary transcripts from their start sites by grey bars, the ribosome binding site as a yellow box and the respective coding sequence from their start sites by green bars.
Figure 2.
Effect of low acidic condition (pH = 2.5) on survival of usp deletion mutants compared to L. monocytogenes EGD-e wild-type.
Δlmo0515 and Δlmo1580 display decreased growth at all observations, while impaired resistance in Δlmo2673 was visible only at 20 min post challenge. Statistically significant differences were identified using a two-tailed Student t test. (*, p<0.05 and **, p<0.005).
Figure 3.
Survival of L. monocytogenes EGD-e wild-type and usp deletion mutants after exposure to H2O2.
As appreciable, resistance of all deletion mutants was strongly impaired, resulting in decreased growth as compared to wild-type strain. Statistically significant differences were identified using a two-tailed Student t test. (*, p<0.05; **, p<0.005, and ***, p<0.0005).
Figure 4.
The intracellular survival of L. monocytogenes EGD-e wild-type and usp deletion mutants was assessed in murine macrophages according to [30].
Intracellular bacteria burden was decreased in macrophages inoculated with Δlmo1580 and Δlmo2673, but no effect was visible for Δlmo0515. Statistically significant differences were identified using a two-tailed Student t test. (*, p<0.05 and **, p<0.005).
Figure 5.
qRT-PCR analysis of lmo0515, lmo1580 and lmo2673 expressed in L. monocytogenes EGD-e.
The graph displays the relative expression of the respective usp gene in the intracellular vs. extracellular setting. High fold changes indicate that the particular gene was higher expressed in intracellularly localized bacteria as compared to expression in bacteria that were grown in BHI. The expression of lmo0515 and lmo2673 is strongly enhanced in intracellular pathogens, while levels of lmo1580 remain relatively constant. This is consistent with the observation made in this study, which shows a strong dependence on lmo2673 and lmo0515 in the murine infection model experiments as compared to extracellular in vitro challenges, while lmo1580 seems to play an important role in both conditions.
Figure 6.
Survival of G. mellonella larvae after inoculation with usp deletion mutants.
As compared to L. monocytogenes EGD-e wild-type, all usp deletion mutants exhibited impaired virulence, resulting in higher survival rates of G. mellonella larvae. Strongest effects were observed for Δlmo1580 and Δlmo2673, while deletion of lmo0515 was not associated with significant increase in survival of G. mellonella. Results represent mean values of at least three independent experiments with a total of 80 larvae per treatment. Statistically significant differences were identified using a two-tailed Student t test. (***, p<0.0005 and ns-not significant).
Figure 7.
Survival rates of intravenously inoculated L. monocytogenes EGD-e or usp deletion mutants in mice livers and spleens.
Survival of mutant bacteria are impaired in both organs, reflecting a role of Usps in listerial resistance and growth in vivo. Statistically significant differences were identified using a two-tailed Student t test. (*, p<0.05).