Transposon mutagenesis as a tool to study the role of hemolysin in the virulence of Listeria monocytogenes

Abstract

The role of hemolysin in the virulence of Listeria monocytogenes was studied by using transposon mutagenesis. The 26-kilobase conjugative transposon Tnl545, originally found in Streptococcus pneumoniae, was transferred to a hemolytic virulent strain of L. monocytogenes. The frequency of transfer was estimated to be about 10-8 per recipient. This allowed us to isolate a nonhemolytic mutant which most likely harbors a single copy of Tn1545. Loss of hemolysin production was associated with loss of virulence. The 50% lethal dose of the mutant was assessed to about 109.6 bacteria per mouse after intravenous challenge. Nonhemolytic bacteria were unable to grow in host tissues and were rapidly eliminated from the spleen and liver of infected mice. Virulence was restored in hemolysin-producing revertant obtained by spontaneous loss of transposon Tnl545. These results strongly suggest that hemolysin is a major virulence factor implicated in the intracellular growth of L. monocytogenes.

Uptake and killing of Listeria monocytogenes by normal human peripheral blood granulocytes and monocytes as measured by flow cytometry and cell sorting

“…A hemolysin-negative mutant of L. monocytogenes strain EGD, CNL85/162 (Hly3), produced by transposon mutagenesis with Tn1545, and a spontaneous revertant which produced LLO and was virulent in mice, CNL85/ 163 (Hly+), were used in these studies. With the exception of LLO in the Hly3 strain, both of these strains carry the full complement of virulence factors including lecithinase activity (PLC) [11]. Bacteria were grown overnight in static culture in trypticase soy broth with yeast extract (Difco, Detroit, MI, USA) to the stationary phase and standardized to an optical density at 550 nm of 0.5.…”

“…Increased susceptibility of gp91 phosx de¢cient mice during the neutrophil-dependent phase demonstrates the importance of oxidative burst in the ability of neutrophils to control the early stages of infection [9]. From the bacterial perspective, listeriolysin O (LLO) allows for escape from the phagocytic vacuole of host macrophages [10] and its deletion markedly decreases virulence in mice [11]. Two phospholipases C (PLC) also in£uence virulence.…”

Cytosolic expression of SecA2 is a prerequisite for long-term protective immunity

“…In contrast to intracellular persistent vacuolar bacteria, L. monocytogenes escapes primary phagocytic vacuoles and rapidly grows inside the cytosol of host cells to establish a productive infectious cycle. L. monocytogenes cytoplasmic escape and multiplication is dependent upon the pore-forming toxin listeriolysin O (LLO) (Gaillard et al, 1986), which acts by dissolving primary phagocytic vacuoles. In this setting, both primary and secondary protective CD8 + T cell responses have been well characterized and suggest to require intracytosolic bacterial growth (Berche et al, 1987).…”

Listeria monocytogenes, a model in infection biology

“…Once in the cytosol, Lm can polymerise actin via its surface protein ActA, and this propels bacteria intracellularly and from one cell to its neighbours, allowing the propagation of the infection into foci without contact with the extracellular milieu (Kocks et al, 1992;Tilney & Portnoy, 1989). LLO and ActA are part of Lm core genome, and their deletion leads to several orders of magnitude loss of virulence in animal models of infection, highlighting the critical importance of Lm intracellular survival in the pathogenesis of listeriosis (Gaillard, Berche, & Sansonetti, 1986;Levraud et al, 2009;Portnoy, Jacks, & Hinrichs, 1988). These two genes are part of Listeria pathogenicity island-1 (LIPI-1) and co-regulated by the transcriptional regulator PrfA, the master regulator of Lm virulence genes (Cossart & Lecuit, 1998).…”