Reduced Host Cell Invasiveness and Oxidative Stress Tolerance in Double and TriplecspGene Family Deletion Mutants ofListeria monocytogenes

Abstract

The cold shock protein (Csp) family comprises small, highly conserved proteins that bind nucleic acids to modulate various bacterial gene expressions. In addition to cold adaptation functions, this group of proteins is thought to facilitate various cellular processes to promote normal growth and stress adaptation responses. Three proteins making up the Listeria monocytogenes Csp family (CspA, CspB, and CspD) promote both cold and osmotic stress adaptation functions in this bacterium. The contribution of these three Csps in the host cell invasion processes of L. monocytogenes was investigated based on human Caco-2 and murine macrophage in vitro cell infection models. The DeltacspB, DeltacspD, DeltacspAB, DeltacspAD, DeltacspBD, and DeltacspABD strains were all significantly impaired in Caco-2 cell invasion compared with the wild-type strain, whereas in the murine macrophage infection assay only, the double (DeltacspBD) and triple (DeltacspABD) csp mutants were also significantly impaired in cell invasion compared with the wild-type strain. The DeltacspBD and DeltacspABD mutants displayed the most severely impaired invasion phenotypes. The invasion ability of these two mutant strains was also further analyzed using cold-stress-exposed organisms. In both cell infection models a significant reduction in invasiveness was observed after cold stress exposure of Listeria organisms. The negative impact of cold stress on subsequent cell invasion ability was, however, more severe in cold-sensitive csp mutants (DeltacspBD and DeltacspABD) compared with the wild type. The impaired macrophage invasion and intracellular growth of DeltacspBD and DeltacspABD also led us to examine oxidative stress resistance capacity in these two mutant strains. Both strains also displayed higher oxidative stress sensitivity relative to the wild-type strain. Our data indicate that besides cold and osmotic stress adaptation roles, Csp family proteins also promote efficient host cell invasion and oxidative stress adaptation processes in L. monocytogenes.

The Contribution of Transcriptomic and Proteomic Analysis in Elucidating Stress Adaptation Responses ofListeria monocytogenes

“…A catalase-deficient L. monocytogenes F2365 mutant was recently described, which is impaired during cold growth (Azizoglu and Kathariou, 2010b). In addition, mutants lacking some csp genes are also characterized by increased peroxide stress sensitivity and reduced host cell infectivity (Loepfe et al, 2010). Moreover, L. monocytogenes mechanisms to combat osmotic stress seem to also involve functions of the cold shock domain family proteins.…”

Cold Shock Proteins Contribute to the Regulation of Listeriolysin O Production inListeria monocytogenes

“…In a previous transcriptome analysis study, it was found that cspA and cspB genes were among other genes activated during host infection (Camejo et al, 2009). Moreover, Csps have been found to facilitate oxidative stress resistance and host cell invasion (Loepfe et al, 2010). L. monocytogenes mutant strains deleted in csp genes (DcspBD and DcspABD) were diminished in host cell infectivity and showed increased oxidative stress susceptibility.…”

Proteomic Analysis Shows That IndividualListeria monocytogenesStrains Use Different Strategies in Response to Gastric Stress

“…Therefore, it seems reasonable to advance the hypothesis that T8 responds to moderate osmolarity (3.5% [w/v] NaCl) in the CM medium with the assistance of the 60-kDa chaperonin (GroEL), ATP synthase F1, b subunit, and the glutamate decarboxylase beta. GroEL has been associated to cold, osmotic, and acid stress responses in L. monocytogenes (Loepfe et al, 2010;Phan-Thanh and Mahouin, 1999;Schmid et al, 2009), whereas F 1 F 0 -ATPase and the glutamate decarboxylase, which is part of the glutamate decarboxylase system (GAD), have been associated with the acid tolerance response (Cotter et al, 2000(Cotter et al, , 2001(Cotter et al, , 2005Karatzas et al, 2010). The glutamate decarboxylase converts extracellular glutamate to c-aminobutyrate (GABA) by consuming an intracellular proton and increasing the internal cellular pH.…”

“…However, adapted T8 cells overproduced the cold shock protein CspLB, suggesting a role for these types of proteins in gastric stress response. Besides their recognized role in cold, osmotic, and oxidative stress protection in Listeria, Csp proteins have been associated with host invasion (Loepfe et al, 2010;Schmid et al, 2009). The higher abundance of SOD by adapted A9 cells suggests that during gastric fluid exposure the bacterial cells experience a secondary oxidative stress, resulting from the exposure to the low pH.…”