Y. pseudotuberculosis has been subclassified into 21 serotypes, which are considered as causative agents of several human and animal diseases (Skurnik, 1999). Y. pseudotuberculosis is the occasional etiologic agent of gastroenteritis, resulting in severe abdominal pain, fever and headache (Putzker et al. 2001).
Y. pseudotuberculosis HtrA protein shares the proteolytic and the two PDZ domains with its orthologues from E. coli (Wessler et al. 2017), Legionella fallonii, and Thermotoga maritime (Hansen and Hilgenfeld, 2013). A regulatory role for the PDZ domains has been demonstrated for HtrA and DegS (Krojer et al. 2010), but currently, there is no evidence for a similar regulation of the Y. pseudotuberculosis HtrA protein. According to in silico modeling, the differences in the amino acid sequence between the Y. pseudotuberculosis HtrA protein and its orthologues from Y. pestis, Chlamydia trachomatis and E. coli (Gloeckl et al. 2012) cluster at the LA loop within the proteolytic domain. The alteration in the coding region introduced in the htrA::Km chimeric gene interrupts the gene at the beginning of the proteolytic domain and encodes an 836 amino acid long polypeptide which is coincident only in the first 70 codons and has less than 25% similarity (at the amino acid level thereafter). Contrary to our expectations, the signal of this large chimeric HtrA::Km polypeptide, or any degradation fragment could not be detected with Abs anti HtrA of S. aureus. As expected, the band corresponding to the size of the HrtA wild-type protein was not present either. Either the chimeric transcript encoding HtrA::Km chimera is not transcribed, it is not translated, it is rapidly degraded at the mRNA or protein level, or all the antigenic determinants are lost due to the mutation. Instead, the HtrA protein is present in the Y. pseudotuberculosis wild type grown at 30, 37 and 42 °C, and under this conditions, it has been reported to have proteolytic activity (Lopes et al. 2009).
In contrast to the mutation by deletion of H. pylori (Zawila-Pawlik et al. 2019), to the mutation by deletion of PDZ domain in Synechocystis reported by Huesgen et al. (Huesgen et al. 2011) and to the site-directed mutagenesis in C. trachomatis reported by Gloeckl et al. (Gloeckl et al. 2012), the insertion of Km resistance cassette into the proteolytic domain coding region of the htrA gene of Y. pseudotuberculosis YPIII wild type strain, here reported, lead to the loss of the HtrA protein signal in the immunoblot assay.
In Salmonella enterica serovar Typhimurium, there is a protein homologue to E. coli HtrA called DegQ, in addition to classic S. enterica HtrA. The protease DegQ has identical activity to HtrA in vitro, but the null mutation of degQ gene does not attenuate the virulence of this strain, whereas the double null mutant in htrA and degQ present virulence attenuation (Farn and Roberts, 2004).
Two proteases of the type HtrA are found in S. aureus and their coding genes are called htrA1 and htrA2. In this case only HtrA1 was found to have a function in the protection against thermal stress, but its protease activity is low or absent, and the role of HtrA1 as a protease may be compensated by a different, yet unidentified protease (Rigoulay, 2005).
Mycobacterium leprae has the gene ML0176, which codes a predicted HtrA-like protease, conserved in other species of mycobacteria (Lopes et al. 2009) and in Mycobacterium tuberculosis. This gene, known as pepD, is directly regulated by the stress-responsive two-component signal transduction system MprAB and indirectly by the so called extracytoplasmic function (ECF) sigma factor, SigE (White et al. 2011).
It has been reported that three serine proteases, HtrA, HhoA (HtrA homologue A) and HhoB (HtrA homologue B), are important for survival of Synechocystis sp. PCC6803 (Huesgen et al. 2011).
In Campylobacter jejuni the HtrA has protease and chaperone activities and participates in the interaction between C. jejuni and mammalian host cells (Bæk et al. 2011).
In Southern blot type hybridizations were performed, using a 2.1 kb fragment as a probe, which contains the htrA gen of Y. pseudotuberculosis, and only one signal was obtained. This last result suggests a unique gene encoding HtrA function in Y. pseudotuberculosis YPIII strain.
In contrast to Y. pseudotuberculosis YPIII wild type strain increase of the temperature strongly reduces the growth of the corresponding 1YPIII htrA− mutant. This result is in agreement with the data reported by Yamamoto (Yamamoto et al. 1996), for a mutant of the gsrA gene of Y. enterocolítica, and with the Williams' results (Williams et al. 2000) for Y. pestis htrA− mutant. In none of these last two reports did the mutant grow at temperatures higher than 39 °C, is agreement with the data reported by Zarzecka for H. pylori (Zarzecka et al. 2019).
In addition, HtrA from M. leprae displayed maximum proteolytic activity at temperatures above 40 °C (Lopes et al. 2009), HtrA from H. pylori showed temperature-dependent oligomer dissociation (Hoy et al. 2013) and DegP was important during high-temperature bacterial growth (Kim and Robert, 2012). In our case, hrtA− mutant showed virtually a total growth arrest after 4 h at 42ºC, and when compared to the wild strain at 12 h, a culture OD difference of 77.82% was observed. As already mentioned, in mutants of the htrA gene of bacteria such as Streptococcus pneumonie (Musa et al. 2004) and S. enterica (Lewis et al. 2009), the growth at 42 °C is attenuated, while the growth at lower temperatures was normal. Therefore, HrtA must be performing a role at 37ºC and 42ºC which is required for normal growth and that cannot be fulfilled by any other chaperones and/or proteases in any of these bacterial species.
In a similar fashion, mutations in the htrA gene of S. pneumonie (Musa et al. 2004), Klebsiella pneumonie (Cortes et al. 2002) and Listeria monocytogenes 10403S (Wonderling et al. 2004) did have no effect in the growth rates at 37ºC, but did reduce the growth and/or survival rates at higher temperatures and in other bacteria as mentioned by Wessler (Wessler et al. 2017). In L. monocytogenes 10403S the sensitivity to high osmolarity was also increased in the mutant.
In the present work, there seem to be an increase in the expression of the HtrA band with the growth temperature in the Y. pseudotuberculosis YPIII wild type strain, (Fig. 3A). This result is in agreement with the increase in the rpoE gene with an increasing growth temperature. This gene encodes a transcriptional regulator which is related to the transcription of the htrA gene in E. coli and is likely to fulfill a similar role in Y. pseudotuberculosis. Yersinia pseudotuberculosis HtrA protein is important for cell growth at 37ºC, it is essential at 42ºC, and its molecular features suggest an unshared involvement in protein folding homeostasis at high temperatures.