Roles of Alternative Sigma Factors in Invasion and Growth Characteristics of Listeria monocytogenes 10403S Into Human Epithelial Colorectal Adenocarcinoma Caco-2 Cell

Junyaluck Rukit; Atsadang Boonmee; Teeratas Kijpornyongpan; Kan Tulsook; József Baranyi; Soraya Chaturongakul

doi:10.3389/fmicb.2022.901484

Roles of Alternative Sigma Factors in Invasion and Growth Characteristics of Listeria monocytogenes 10403S Into Human Epithelial Colorectal Adenocarcinoma Caco-2 Cell

Front Microbiol. 2022 Jul 14:13:901484. doi: 10.3389/fmicb.2022.901484. eCollection 2022.

Authors

Junyaluck Rukit¹, Atsadang Boonmee², Teeratas Kijpornyongpan³, Kan Tulsook⁴, József Baranyi⁵, Soraya Chaturongakul^{1

6}

Affiliations

¹ Department of Microbiology, Faculty of Science, Mahidol University, Bangkok, Thailand.
² Department of Microbiology, Faculty of Science, Chulalongkorn University, Bangkok, Thailand.
³ Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN, United States.
⁴ National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Pathum Thani, Thailand.
⁵ Institute of Nutrition, University of Debrecen, Debrecen, Hungary.
⁶ Molecular Medical Biosciences Cluster, Institute of Molecular Biosciences, Mahidol University, Nakhon Pathom, Thailand.

Abstract

Listeria monocytogenes is a Gram-positive facultative intracellular bacterium with a broad host range. With its housekeeping sigma factor and four alternative ones (namely SigB, SigC, SigH, and SigL), L. monocytogenes can express genes in response to changing environments. However, the roles of these sigma factors in intracellular survival are still unclear. The objectives of this study were to characterize the role of each alternative σ factor on L. monocytogenes invasion and growth inside human epithelial colorectal adenocarcinoma Caco-2 cells. We used L. monocytogenes 10403S wild type and its 15 alternative sigma factor deletion mutants at a multiplicity of infection of 100 and 1 in invasion and intracellular growth assays in the Caco-2 cells, respectively. At 1.5, 2, 4, 6, 8, 10, and 12 h post-infection, Caco-2 cells were lysed, and intracellular L. monocytogenes were enumerated on brain-heart infusion agar. Colony-forming and growth rates were compared among strains. The results from phenotypic characterization confirmed that (i) SigB is the key factor for L. monocytogenes invasion and (ii) having only SigA (ΔsigBCHL strain) is sufficient to invade and multiply in the host cell at similar levels as the wild type. Our previous study suggested the negative role of SigL in bile stress response. In this study, we have shown that additional deletion of the rpoN (or sigL) gene to ΔsigB, ΔsigC, or ΔsigH could restore the impaired invasion efficiencies of the single mutant, suggesting the absence of SigL could enhance host invasion. Therefore, we further investigated the role of SigL during extracellular and intracellular life cycles. Using RNA sequencing, we identified 118 and 16 SigL-dependent genes during the extracellular and intracellular life cycles, respectively. The sigL gene itself was induced by fivefolds prior to the invasion, and 5.3 folds during Caco-2 infection, further suggesting the role of SigL in intracellular growth.

Keywords: Caco-2 cell; Listeria monocytogenes; RNA sequencing; invasion; transcriptomics.