On the role of cell chaining in the attenuation of a Listeria monocytogenes divIVA mutant
Listeria monocytogenes is a facultative intracellular human pathogen capable of invading non-phagocytic host cells, replicating within their cytosol, and spreading directly from cell to cell. These processes are mediated by specialized virulence factors but also depend on the DivIVA protein. DivIVA aids in the secretion of peptidoglycan-degrading autolysins in a process that requires the accessory secretion ATPase SecA2, thereby promoting daughter cell separation following cytokinesis. Consequently, a {Delta}divIVA mutant forms elongated chains of unseparated daughter cells, which may explain its attenuated virulence. To further explore the role of cell chaining for attenuation, we here investigated how different morphological aberrations affect the virulence of L. monocytogenes. To this end, we generated coccoid mreB and filamentous ezrA mutants, and compared them to the {Delta}divIVA mutant in different in vitro infection assays. Coccoid or filamentous morphologies did not impair host cell invasion or intracellular replication, unlike cell chaining of the {Delta}divIVA mutant. Introduction of a hyperactive allele of the PrfA virulence regulator, resulting in constitutive overexpression of virulence genes, was sufficient to restore the invasion defect of the {Delta}divIVA mutant, despite its pronounced cell chaining phenotype, but did not recover intracellular replication. We isolated suppressors of the {Delta}divIVA mutant carrying mutations in secA2, which likely enhance the SecA2 ATPase activity. In these suppressors, autolysin secretion, daughter cell separation, and invasion were fully restored, and intracellular replication was partially recovered. Thus, maintaining normal rod-shaped morphology plays a minor role in L. monocytogenes pathogenesis. Instead, virulence attenuation in the {Delta}divIVA mutant is better explained by distortions in PrfA- and SecA2-dependent processes. Author SummaryListeria monocytogenes infects humans by entering host cells, replicating intracellularly, and spreading from infected cells to neighboring cells. The bacterium is rod-shaped and normally occurs as single or double cells that can efficiently complete all stages of infection. However, a class of virulence-attenuated L. monocytogenes mutants forms long cell chains, suggesting a link between cell morphology and virulence. One such mutant lacks the cell division protein DivIVA and is severely impaired in infection, as it neither invades host cells, replicates in the cytoplasm nor spreads from cell to cell. To determine whether virulence attenuation is caused by cell chaining itself or by additional functional defects, we compared the virulence of the divIVA mutant with that of mutants exhibiting other morphological abnormalities. In addition, we isolated suppressor mutations that restore normal cell separation in the divIVA mutant and examined their effects on virulence. Our results show that cell morphology per se does not generally determine listerial virulence. Instead, they indicate that specific virulence-related functions are impaired in chain-forming L. monocytogenes mutants. These findings advance our understanding of the virulence defects associated with cell chaining and provide a basis for identifying their real underlying molecular causes.