Characterization of Salmonella pathogenicity island 1 type III secretion-dependent hemolytic activity in Salmonella enterica serovar Typhimurium

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Abstract

A number of bacteria that are pathogenic for animals and plants possess a type III secretion system (TTSS) to translocate virulence-associated proteins into host cells. In several bacteria, it has been reported that the TTSS is correlated with an ability to cause contact-dependent hemolysis in vitro. Here, we showed that the Salmonella enterica serovar Typhimurium strain SL1344 exhibited Salmonella pathogenicity island 1 type III secretion-dependent, contact-mediated, hemolytic activity. Mutations with a single deletion in genes encoding putative pore-forming proteins, SipB and SipC, secreted by the TTSS abolished hemolytic activity. In addition, the osmoprotection studies revealed that molecules larger than PEG2000 conferred significant protection against lysis induced by Salmonella. These results indicate that the hemolysis generated by Salmonella is due to the formation of pores within the erythrocyte membrane.

Introduction

The type III secretion system (TTSS) is widespread in Gram-negative pathogens including Salmonella, Shigella, Yersinia, enteropathogenic Escherichia coli (EPEC) and Bordetella and in plant pathogens such as Pseudomonas syringae and Erwinia chrysanthemi[1]. TTSS is a complex organelle composed of more than 20 different protein subunits. These proteins are organized into a supramolecular complex, spanning inner and outer membranes of the bacterial cell with an external needle structure [2]. The enteric pathogenic bacteria, Salmonella enterica serovar Typhimurium, possess two distinct TTSSs encoded within Salmonella pathogenicity island 1 and 2 (SPI-1 and SPI-2), which are required for different stages of infection. SPI-1 TTSS is required to initiate intestinal infection, while SPI-2 TTSS appears to be essential for the establishment of systemic infection [3], [4], [5], [6]. SPI-1 encodes all of the proteins necessary to assemble a complex type III secretion apparatus and some of the secreted effector proteins. Functional SPI-1 TTSS requires close contact between infecting bacteria and host cell to secrete and deliver effector proteins to the cytosol of the host cell [7], [8].

In Salmonella, translocation of effector proteins across the host cell membrane requires SipB, SipC, and SipD proteins (referred to as Ssp proteins). Secreted SipB and SipC have been shown to integrate into the host cell membrane and, based on homology with IpaB and IpaC proteins in Shigella, YopB and YopD proteins in Yersinia, and EspB and EspD proteins in EPEC, BopB protein in Bordetella, are thought to be components of the translocation apparatus. All these secreted proteins have been shown to be delivered to the host cell membrane, where they form a pore complex [9], [10], [11], [12], [13], [14], [15], [16], [17]. In all of these bacteria, injection of proteins into host cells by TTSS has been tightly correlated with their ability to cause contact-dependent hemolysis of red blood cells (RBCs) in vitro [18], [19], [20], while in the case of EPEC, intimate contact between bacteria and RBCs is not required for TTSS-mediated hemolysis [21]. However, it has been shown that Salmonella strains could not induce significant lysis of erythrocytes under the same set of conditions by which S. flexneri cause contact hemolysis [18], [22], [23].

Although the TTSS apparatus and translocation proteins are structurally and functionally conserved among several bacterial species, conditions that result in the optimal expression of TTSS phenotypes are different among bacterial strains. In Salmonella, expression of SPI-1 TTSS is regulated in response to environmental and physiological cues, such as osmolarity, oxygen, and pH [24], [25]. Therefore, we have examined the effect of different conditions on the ability of the Salmonella serovar Typhimurium to react with RBCs. Here, we found that Salmonella exhibited a SPI-1 dependent, contact-mediated hemolytic activity requiring the membrane inserting molecules, SipB and SipC. In addition, an osmoprotection assay revealed that SPI-1 TTSS exhibited pore-forming activity in RBCs, and the size of the pores introduced into RBCs by SPI-1 TTSS was estimated to be 3.5 nm.

Section snippets

Salmonella serovar Typhimurium possess SPI-1 TTSS-mediated hemolytic activity

To investigate the interaction of Salmonella with RBCs, we have employed an RBC monolayer model of infection [26]. Monolayers of sheep red blood cells (SRBCs) attached to polylysine-coated glass coverslips were infected with Salmonella serovar Typhimurium wild-type strain SL1344 and a type III secretion-deficient mutant SB136 (invA::kan). Bacteria grown in BHI medium under low-O2 conditions to promote maximal expression of SPI-1 TTSS were used for RBC infection. After centrifugation to obtain

Discussion

The experiments presented here were undertaken to characterize the previously unreported contact-hemolytic activity of Salmonella serovar Typhimurium. In this report, we have detected the hemolytic activity of Salmonella by adapting the growth conditions which are required for full expression of the SPI-1 TTSS. We have shown that, in order to obtain maximal activity of Salmonella-induced hemolysis, bacteria are grown as static culture in BHI broth until OD600=0.8. These are apparently different

Bacterial strains, plasmids, primers and growth conditions

The bacterial strains used in this study are listed in Table 1. Bacteria were routinely grown at 37 °C in Luria-Bertani (LB) broth with shaking, unless otherwise stated. For contact hemolysis assays, Salmonella and Shigella strains were cultured in brain-heart infusion (BHI) medium (Difco Laboratories Inc., Detroit, MI, USA). Phage P22-mediated transductions were performed as described previously [33]. When appropriate, antibiotics were used at the following concentrations: ampicillin at 100 μg

Acknowledgements

We would like to thank B.L. Wanner for the gift of plasmids and strains for the Red disruption system, Akio Abe and Asaomi Kuwae for their helpful suggestions, and Sachiko Tsuruoka, Ryoko Ito, and Satomi Sugiura for their technical assistance. This work was supported in part by a Grant-in-aid for Exploratory Research (15659105) and by a 21st Century COE Program Grant from the Japanese Ministry of Education, Culture, Sports, Sciences, and Technology (MEXT).

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