Seong Ho Bang

CadC Has a Global Translational Effect during Acid Adaptation in Salmonella enterica Serovar Typhimurium

In Salmonella enterica serovar Typhimurium, the membrane-localized CadC is a transcriptional activator of the cadBA operon, which contributes to the acid tolerance response. Unlike in Escherichia coli, in which transcription of cadC is constitutive, in S. enterica serovar Typhimurium cadC expression is induced by low pH and lysine. Inactivation of cadC suppresses the acid-sensitive phenotype of a cadA mutation, suggesting the existence of other CadC-dependent genes in addition to the cadBA operon. Using a proteomic approach, we identified 8 of the putative CadC-induced proteins and 15 of the putative CadC-repressed proteins. The former include porin proteins OmpC and OmpF. The latter include proteins involved in glycolysis, energy production, and stress tolerance. To better understand the altered levels of OmpC and OmpF, we compared expression of ompR in S. enterica serovar Typhimurium wild-type and cadC mutant strains and determined that CadC exerted a negative influence on ompR transcription. Taken together, our findings strongly suggest that CadC may be a global regulator involved in the OmpR regulatory system during acid adaptation.

Role of Salmonella Pathogenicity Island 1 Protein IacP in Salmonella enterica Serovar Typhimurium Pathogenesis

ABSTRACT Gram-negative bacteria, including Salmonella enterica serovar Typhimurium, exploit type III secretion systems (T3SSs) through which virulence proteins are delivered into the host cytosol to reinforce invasive and replicative niches in their host. Although many secreted effector proteins and membrane-bound structural proteins in the T3SS have been characterized, the functions of many cytoplasmic proteins still remain unknown. In this study, we found that IacP, encoded by Salmonella pathogenicity island 1, was important for nonphagocytic cell invasion and bacterial virulence. When the iacP gene was deleted from several Salmonella serovar Typhimurium strains, the invasion into INT-407 epithelial cells was significantly decreased compared to that of their parental strains, and retarded rearrangements of actin fibers were observed for the iacP mutant-infected cells. Although IacP had no effect on the secretion of type III translocon proteins, the levels of secretion of the effector proteins SopB, SopA, and SopD into the culture medium were decreased in the iacP mutant. In a mouse infection model, mice infected with the iacP mutant exhibited alleviated pathological signs in the intestine and survived longer than did wild-type-infected mice. Taken together, IacP plays a key role in Salmonella virulence by regulating the translocation of T3SS effector proteins.

Molecular characterization of the InvE regulator in the secretion of type III secretion translocases in Salmonella enterica serovar Typhimurium.

The type III secretion systems (T3SSs) are exploited by many Gram-negative pathogenic bacteria to deliver a set of effector proteins into the host cytosol during cell entry. The T3SS of Salmonella enterica serovar Typhimurium is composed of more than 20 proteins that constitute the membrane-associated base, the needle and the tip complex at the distal end of the T3SS needle. Membrane docking and piercing between the T3SS and host cells is followed by the secretion of effector proteins. Therefore, a secretion hierarchy among the substrates of the T3SS is required. The secretion of the pore-forming translocase proteins SipB, SipC and SipD is controlled by the T3SS regulator protein, InvE. During an attempt to identify the regions of InvE that are involved in T3SS regulation, it was observed that the secretion of SipB, SipC and SipD was inhibited when the C-terminal 52 amino acids were removed from InvE. In addition, InvE derivatives lacking the N-terminal 30 and 100 residues were unable to secrete translocases into the culture medium. Interestingly, in the absence of the N-terminal 180 residues of InvE, SipD is unstable, resulting in the hypersecretion of SipB. We also found that both the type III secretion signals of SipB and SptP were functionally interchangeable with the first 30 amino acids of InvE, which could allow the secretion of a reporter protein. These results indicate that InvE may have two functional domains responsible for regulating the secretion of translocases: an N-terminal secretion signal and a C-terminal regulatory domain.

N-terminal residues of SipB are required for its surface localization on Salmonella enterica serovar Typhimurium

SipB, one of the invasion proteins encoded in Salmonella pathogenicity island 1 (SPI-1), is known to be secreted outside the cell, where it functions as a translocon by assembling into a host-cell plasma membrane-integral structure. Here, we confirmed that wild-type SipB could be localized to the bacterial outer membrane, and further showed that its localization was dependent on extracellular secretion, and was independent of the presence of the SipD protein. Proteinase K susceptibility and immunofluorescence assays indicated that SipB was not incorporated into the outer membrane, but rather was displayed on the bacterial surface. Finally, mutation studies revealed that the N-terminal 100-140 aa (especially amino acids 135-138) of SipB were required for its localization on the bacterial outer membrane.

A phosphotransferase system permease is a novel component of CadC signaling in Salmonella enterica

In Salmonella enterica serovar Typhimurium, proteolytic cleavage of the membrane-bound transcriptional regulator CadC acts as a switch to activate genes of the lysine decarboxylase system in response to low pH and lysine signals. To identify the genetic factors required for the proteolytic activation of CadC, we performed genome-wide random mutagenesis. We show that a phosphotransferase system (PTS) permease STM4538 acts as a positive modulator of CadC function. The transposon insertion in STM4538 reduces the expression of the CadC target operon cadBA under permissive conditions. In addition, deletional inactivation of STM4538 in the wild-type background leads to the impaired proteolytic cleavage of CadC. We also show that only the low pH signal is involved in the proteolytic processing of CadC, but the lysine signal plays a role in the repression of the lysP gene encoding a lysine-specific permease, which negatively controls expression of the cadBA operon. Our data suggest that the PTS permease STM4538 affects proteolytic processing, which is a necessary but not sufficient step for CadC activation, rendering CadC able to activate target genes.