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Listeria monocytogenes , a Gram-positive food-borne human pathogen, is able to grow at temperatures close to 0°C and is thus of great concern for the food industry. In this work, we investigated the physiological role of one DExD-box RNA helicase in Listeria monocytogenes . The RNA helicase Lmo1722 was required for optimal growth at low temperatures, whereas it was dispensable at 37°C. A Δ lmo1722 strain was less motile due to downregulation of the major subunit of the flagellum, FlaA, caused by decreased flaA expression. By ribosomal fractionation experiments, it was observed that Lmo1722 was mainly associated with the 50S subunit of the ribosome. Absence of Lmo1722 decreased the fraction of 50S ribosomal subunits and mature 70S ribosomes and affected the processing of the 23S precursor rRNA. The ribosomal profile could be restored to wild-type levels in a Δ lmo1722 strain expressing Lmo1722. Interestingly, the C-terminal part of Lmo1722 was redundant for low-temperature growth, motility, 23S rRNA processing, and appropriate ribosomal maturation. However, Lmo1722 lacking the C terminus showed a reduced affinity for the 50S and 70S fractions, suggesting that the C terminus is important for proper guidance of Lmo1722 to the 50S subunit. Taken together, our results show that the Listeria RNA helicase Lmo1722 is essential for growth at low temperatures, motility, and rRNA processing and is important for ribosomal maturation, being associated mainly with the 50S subunit of the ribosome.

RNA-helicases are proteins required for the unwinding of occluding secondary RNA structures, especially at low temperatures. In this work, we have deleted all 4 DExD-box RNA helicases in various combinations in the Gram-positive pathogen Listeria monocytogenes. Our results show that 3 out of 4 RNA-helicases were important for growth at low temperatures, whereas the effect was less prominent at 37°C. Over-expression of one RNA-helicase, Lmo1450, was able to overcome the reduced growth of the quadruple mutant strain at temperatures above 26°C, but not at lower temperatures. The maturation of ribosomes was affected in different degrees in the various strains at 20°C, whereas the effect was marginal at 37°C. This was accompanied by an increased level of immature 23S rRNA precursors in some of the RNA-helicase mutants at low temperatures. Although the expression of the PrfA regulated virulence factors ActA and LLO decreased in the quadruple mutant strain, this strain showed a slightly increased infection ability. Interestingly, even though the level of the virulence factor LLO was decreased in the quadruple mutant strain as compared with the wild-type strain, the hly-transcript (encoding LLO) was increased. Hence, our results could suggest a role for the RNA-helicases during translation. In this work, we show that DExD-box RNA-helicases are involved in bacterial virulence gene-expression and infection of eukaryotic cells.

RNA helicases have been shown to be important for the function of RNA molecules at several levels, although their putative involvement in microbial pathogenesis has remained elusive. We have previously shown that Listeria monocytogenes DExD-box RNA helicases are important for bacterial growth, motility, ribosomal maturation, and rRNA processing. We assessed the importance of the RNA helicase Lmo0866 (here named CshA) for expression of virulence traits. We observed a reduction in hemolytic activity in a strain lacking CshA compared to the wild type. This phenomenon was less evident in strains lacking other RNA helicases. The reduced hemolysis was accompanied by lower expression of major listerial virulence factors in the ΔcshA strain, mainly listeriolysin O, but also to some degree the actin polymerizing factor ActA. Reduced expression of these virulence factors in the strain lacking CshA did not, however, correlate with a decreased level of the virulence regulator PrfA. When combining the ΔcshA knockout with a mutation creating a constitutively active PrfA protein (PrfA*), the effect of the ΔcshA knockout on LLO expression was negated. These data suggest a role for the RNA helicase CshA in posttranslational activation of PrfA. Surprisingly, although the expression of several virulence factors was reduced, the ΔcshA strain did not demonstrate any reduced ability to infect nonphagocytic cells compared to the wild-type strain.