Salmonella enterica Serovar Typhimurium HtrA: regulation of expression and role of the chaperone and protease activities during infection

Abstract

HtrA is a bifunctional stress protein required by many bacterial pathogens to successfully cause infection. Salmonella enterica serovar Typhimurium (S. Typhimurium) htrA mutants are defective in intramacrophage survival and are highly attenuated in mice. Transcription of htrA in Escherichia coli is governed by a single promoter that is dependent on s E (RpoE). S. Typhimurium htrA also possesses a s E -dependent promoter; however, we found that the absence of s E had little effect on production of HtrA by S. Typhimurium. This suggests that additional promoters control expression of htrA in S. Typhimurium. We identified three S. Typhimurium htrA promoters. Only the most proximal promoter, htrAp3, was s E dependent. The other promoters, htrAp1 and htrAp2, are probably recognized by the principal sigma factor s 70 . These two promoters were constitutively expressed but were also slightly induced by heat shock. Thus expression of htrA is different in S. Typhimurium and E. coli. The role of HtrA is to deal with misfolded/damaged proteins in the periplasm. It can do this either by degrading (protease activity) or folding/capturing (chaperone/sequestering, C/S, activity) the aberrant protein. We investigated which of these functions are important to S. Typhimurium in vitro and in vivo. Point or deletion mutants of htrA that encode variant HtrA molecules have been used in previous studies to investigate the role of different regions of HtrA in C/S and protease activity. These htrA variants were placed under the control of the S. Typhimurium htrAP123 promoters and expressed in a S. Typhimurium htrA mutant, GVB1343. Both wild-type HtrA and HtrA (HtrA S210A) lacking protease activity enabled GVB1343 to grow at high temperature (46 6C). Both molecules also significantly enhanced the growth/survival of GVB1343 in the liver and spleen of mice during infection. However, expression of wild-type HtrA enabled GVB1343 to grow to much higher levels than expression of HtrA S210A. Thus both the protease and C/S functions of HtrA operate in vivo during infection but the protease function is probably more important. Absence of either PDZ domain completely abolished the ability of HtrA to complement the growth defects of GVB1343 in vitro or in vivo.

Different Contributions of HtrA Protease and Chaperone Activities to Campylobacter jejuni Stress Tolerance and Physiology

“…Consistent with findings in E. coli (63), the protease activity of C. jejuni HtrA is more efficient at high temperature, while the chaperone activity of HtrA S197A is slightly reduced at 44°C. Interestingly, we found that chaperone activity alone is sufficient for growth of C. jejuni at high temperature, while E. coli and Salmonella require plasmid-mediated overexpression of protease-negative HtrA to suppress the temperature-sensitive phenotype of an htrA deletion mutant (34,61), suggesting that the chaperone activity of C. jejuni is more efficient. Interestingly, only when the temperature and oxygen stresses are combined is protease activity essential for growth of C. jejuni.…”

“…However, despite a detailed mechanistic insight into HtrA function (26,31,32), only few studies have addressed how the individual HtrA activities contribute to stress tolerance in bacteria. These studies have shown that plasmid-mediated expression of an HtrA mutant protein that lacks protease activity but retains chaperone activity allows growth of E. coli and Salmonella htrA deletion mutants at high temperature (34,61,63). Such experiments indicate that the protease activity may be dispensable if the HtrA chaperone activity is present in elevated amounts; however, they do not show how each of these activities contributes to stress tolerance in bacteria.…”

“…We previously showed that HtrA is required for growth of C. jejuni at elevated oxygen tensions and under conditions that induce protein misfolding, such as high temperature and puromycin (6). While bacterial htrA mutants show different stress-sensitive phenotypes, all htrA mutants examined so far are sensitive to high temperature (9,10,34,35,48,69). In addition, several studies have suggested an important role of HtrA in virulence of C. jejuni, as an htrA mutant invades epithelial cells (6,44) and kills infected Galleria mellonella larvae less efficiently than the wild type (8).…”

Proteolytic activation of Chlamydia trachomatis HTRA is mediated by PDZ1 domain interactions with protease domain loops L3 and LC and beta strand β5

“…Chronic infection can lead to serious medical conditions, including infertility, ectopic pregnancy, epididymitis, and pelvic inflammatory disease [2], but the pathogenic mechanisms are poorly understood. Recent studies identified the protease/chaperone CtHtrA as a potential virulence factor as it is upregulated during C. trachomatis disease models [3] and implicated in the pathogenesis of several other bacteria, including Legionella pneumophila, Salmonella enterica, and Helicobacter pylori [4][5][6]. HtrA (high temperature requirement A; also known as DegP) has been extensively studied in Escherichia coli, where it has been characterized as both a serine protease and a chaperone that functions in the maintenance of periplasmic integrity through the degrading, refolding, and chaperoning of protein substrates [7][8][9].…”

HtrA, a Temperature- and Stationary Phase-Activated Protease Involved in Maturation of a Key Microbial Virulence Determinant, Facilitates Borrelia burgdorferi Infection in Mammalian Hosts

“…HtrA proteases function primarily in protein homeostasis and quality control, acting as proteases and chaperones that stabilize specific proteins and modulate signaling pathways (12). Similarly, HtrA proteases have been reported to play an essential role in the virulence of a number of pathogens, such as Mycobacterium tuberculosis (15), Salmonella enterica (16,17), Helicobacter pylori (18), Streptococcus pneumoniae (19), Bacillus anthracis (20), and Staphylococcus aureus (21), and have also been associated with several human diseases (21)(22)(23). While the predominant mechanism of loss of virulence lies in the substrate proteins that are stabilized or processed by HtrA, a direct role for HtrA in pathogen invasiveness has also been reported (18,24).…”