The Yersinia enterocolitica Ysa type III secretion system is expressed during infections both in vitro and in vivo

Abstract

Yersinia enterocolitica biovar 1B maintains two type III secretion systems (T3SS) that are involved in pathogenesis, the plasmid encoded Ysc T3SS and the chromosomally encoded Ysa T3SS. In vitro, the Ysa T3SS has been shown to be expressed only at 26°C in a high-nutrient medium containing an exceptionally high concentration of salt – an artificial condition that provides no clear insight on the nature of signal that Y. enterocolitica responds to in a host. However, previous research has indicated that the Ysa system plays a role in the colonization of gastrointestinal tissues of mice. In this study, a series of Ysa promoter fusions to green fluorescent protein gene (gfp) were created to analyze the expression of this T3SS during infection. Using reporter strains, infections were carried out in vitro using HeLa cells and in vivo using the mouse model of yersiniosis. Expression of green fluorescent protein (GFP) was measured from the promoters of yspP (encoding a secreted effector protein) and orf6 (encoding a structural component of the T3SS apparatus) in vitro and in vivo. During the infection of HeLa cells GFP intensity was measured by fluorescence microscopy, while during murine infections GFP expression in tissues was measured by flow cytometry. These approaches, combined with quantification of yspP mRNA transcripts by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), demonstrate that the Ysa system is expressed in vitro in a contact-dependent manner, and is expressed in vivo during infection of mice.

Transcriptomic Analysis of Yersinia enterocolitica Biovar 1B Infecting Murine Macrophages Reveals New Mechanisms of Extracellular and Intracellular Survival

“…We also demonstrate, for the first time in mammalian cells, that mutants of the Ysa T3SS exhibit a decreased ability to survive within murine macrophages. Upon combining the results of previous studies (31,69) with our present findings, it seems apparent the Ysa system does in fact play a role during the course of an infection, but only when the bacteria have been internalized. This would explain the phenotype of a defect in the initial colonization of mice, since bacteria expressing the system are better able to avoid being killed by the macrophages and are therefore able to more rapidly colonize the terminal ileum and Peyer's patches in the first 24 h after infection.…”

“…Previous work has demonstrated that this secondary T3SS is required for full virulence of the bacterium in a mouse model of infection, but only at very early time points (69). Despite this finding, we recently demonstrated that the Ysa system is expressed throughout the course of murine infections in a contact-dependent manner in every tissue examined (31). Initial studies found the Ysa T3SS to be expressed in vitro only at 26°C in nutrient-rich media containing Ͼ150 mmol/liter salt (NaCl or KCl) (69), conditions that would not suggest a role in the infection of a mammalian host.…”

“…Although the system has been shown to be expressed throughout the infection of mice (31), it has been shown to be important only within the first 24 h after infection (69). It was also recently found to play an important role in the intracellular survival of the bacteria in Drosophila S2 cells (70), although Y. enterocolitica is not known to infect insects.…”

“…Strain GY4478 (JB580 pYVϪ) (27) was used as the parent strain for making insertion mutants of the Yts2 T2SS and Tad pilus in order to prevent the rapid killing of host cells by the Ysc T3SS. Plasmid pEP185.2 (24) was inserted into genes thought to be critical to the function of the Yts2 T2SS and Tad pilus (28)(29)(30), essentially as previously described (31). Briefly, 300-to 500-bp fragments of the genes yts2D (primer pair yts2D-F [5=-TCT AGA CCT ATA GTA GGA TAT GCG GAG AAC-3=] and yts2D-R [5=-TCT AGA CTG GCG TAG TAA CGG AGC-3=]) and tadA (primer pair tadA-F [5=-TCT AGA GCT CGC CAG TAT TGA TAT AGA TC-3=] and tadA-R [5=-TCT AGA CGT CCA ATG CAA TAG GGC-3=]) were PCR amplified and cloned into pCR-BluntII-TOPO (Life Technologies).…”

The YsrS Paralog DygS Has the Capacity To Activate Expression of the Yersinia enterocolitica Ysa Type III Secretion System

“…In addition, this T3SS may also provide a survival benefit in a mammalian host environment. During mouse infections, activation of ysa expression was evident in intestinal and lymphatic tissue by 48 h postinfection (18). Upregulation of ysa genes was also detected from intracellular Y. enterocolitica during mouse macrophage tissue culture infection (19).…”