Shem-Tov, R. | bioRxiv Science

bioRxiv2026

Genome degradation in the c-di-GMP signaling pathways and serovar-specific YhjH-YciG interactions rewired motility regulation in Salmonella Paratyphi A

Salmonella enterica serovars Typhimurium (STM) and Paratyphi A (SPA) cause clinically distinct diseases, yet the molecular bases for their different lifestyles remain incompletely understood. Genome degradation, a hallmark of typhoidal Salmonella, results in extensive pseudogenization across multiple functional pathways. Here, we investigate how such gene inactivation rewires cyclic-di-GMP (c-di-GMP) signaling and flagellar motility regulation in SPA vs. STM. We show that YhjH, a conserved phosphodiesterase (PDE), is required for motility in STM but not in SPA, despite retaining PDE activity in both serovars. We demonstrate that this functional divergence is caused by pseudogenization of ycgR in SPA, which truncates the flagellar brake protein YcgR to a nonfunctional peptide, severing the link between c-di-GMP levels and flagellar motor control. Site directed mutagenesis in the YhjH active site and ectopic expression of intact YcgR from STM that restored YhjH-dependent motility regulation in SPA, confirmed this molecular mechanism. Additionally, using a bacterial two-hybrid (BACTH) genetic screen, we identified a serovar-specific interaction between YhjH and the general stress protein YciG in SPA, but not in STM. Computational RNA folding analysis predicted substantial differences in mRNA secondary structure and stability between the SPA and STM yhjH alleles, suggesting a potential role for synonymous mutations in this serovar-specific interaction. Together, these findings reveal how genome degradation can rewire regulatory networks, uncovering a fundamental difference in motility control between typhoidal and non-typhoidal Salmonella and suggest that these differences allow SPA motility under conditions that suppress motility in STM. IMPORTANCESalmonella enterica serovars Typhimurium (STM) and Paratyphi A (SPA) cause fundamentally different diseases in humans, yet the molecular basis for their distinct lifestyles and pathogenicity remains poorly understood. Genome degradation is a hallmark of typhoidal Salmonella, but its functional consequences for regulatory networks are largely unexplored. Here, we demonstrate that pseudogenization of ycgR in SPA dismantles c-di-GMP-mediated flagellar motor control, liberating SPA from an inhibitory brake that suppresses motility in STM. Additionally, we uncover a serovar-specific interaction between the phosphodiesterase YhjH and the general stress protein YciG in SPA, demonstrating that genome degradation can drive regulatory network rewiring beyond simple gene loss. These differences in motility regulation may facilitate the systemic pathogenesis and unique lifestyle of SPA.

microbiology↗

bioRxiv2025

HilE mediates motility thermoregulation in typhoidal Salmonella serovars at elevated physiological temperatures

Salmonella enterica is a diverse bacterial pathogen consisting of both typhoidal and nontyphoidal clinically distinct serovars. While typhoidal serovars cause in humans a systemic life-threatening enteric fever, nontyphoidal Salmonella (NTS) usually provoke a localized self-limiting gastroenteritis. Factors responsible for the different diseases caused by distinct Salmonella serovars are still poorly understood. Here, we show that at elevated physiological temperature, manifested during enteric fever (39-40{degrees}C), the transcription of the flagellar regulon, its protein translation, and flagella-mediated motility are all repressed in the typhoidal serovar, S. Paratyphi A (SPA). In contrast, the NTS representative serovar, S. Typhimurium, maintains similar or even higher levels of flagellar genes transcription, translation, and motility at 40{degrees}C relative to 37{degrees}C. By using a temperature-responsive flagellar reporter system in conjunction with a dense transposon mutagenesis screen we found that under elevated temperature, HilE negatively regulates SPA motility in a HilD-dependent manner. Since HilD is required for the transcriptional activation of FlhDC, the master regulator of the Salmonella flagellar-chemotaxis regulon, null deletion of hilE leads to motility upregulation at elevated temperature and the loss of motility thermoregulation in SPA. Interestingly, the absence of HilE also leads to a hyper-uptake of SPA by THP- 1 human macrophages at 40{degrees}C, but not at 37{degrees}C. Moreover, we show that a HilE-mediated motility thermoregulation is common to other typhoidal serovars, including S. Typhi and S. Sendai, but not to S. Paratyphi B, nor to various NTS serovars. Based on these results, we propose that HilE plays a unique role in motility thermoregulation in typhoidal Salmonella in a way that may restrain systemic dissemination of the pathogen via professional phagocytes, during the acute phase of enteric fever. AUTHOR SUMMARYDespite high genetic similarity, typhoidal and nontyphoidal Salmonella (NTS) strains of the single species Salmonella enterica cause in humans different diseases manifested as life-threatening enteric fever and short-term gastroenteritis, respectively. Currently, we are still ignorant about bacterial factors shaping the different lifestyles of typhoidal vs. NTS strains. Here we characterized differences in the regulation of Salmonella motility, which is an important virulence-associated phenotype, in response to changes in temperature, between typhoidal and NTS. We found that at elevated temperature, equivalent to the body temperature during enteric fever (39-40{degrees}C), the motility of typhoidal Salmonella, but not that of NTS is strongly repressed, by the negative regulator HilE in a HilD-dependent manner. Moreover, we demonstrate that HilE plays a previously unknown role in the interaction of S. Paratyphi A with phagocytic cells, as the absence of HilE caused enhanced uptake of this pathogen by human macrophages at elevated physiological temperature, but not at 37{degrees}C. Since motility thermoregulation by HilE was found in three different typhoidal serovars, but not in NTS, we hypothesize that motility regulation affects the interactions of Salmonella with its host and differences in its regulation contribute to the distinct pathogenicity of typhoidal vs. NTS strains.

microbiology↗