Abstract/Summary

Salmonella enterica serovar Enteritidis (SE) is an important Salmonella serotype that causes significant human infection through its contamination of poultry meat and eggs. Identifying processes that confer resistance to egg white (EW) might explain, and help combat, the ability of SE to survive in the harsh conditions of EW. The study described herein builds upon pervious work which shows that a set of hexonate/hexuronate (Hex) utilisation genes (dgoRKADT, uxuAB-uxaC and SEN1433-6 genes) are the most strongly induced when SE is exposed to EW. This observation is a surprise since no evidence for the presence of Hex substrates in EW is available, and these Hex utilisation (hex) genes are not know to have any role in EW survival. To study the regulation of the above ‘hex’ genes in response to EW, lacZ transcriptional fusions were generated to each of the potential promoter regions. The resulting transcriptional fusion data showed that seven of the fusions have activity markedly above that of the vector control, but two (dgoT; SEN2979) have weak activity, suggesting no promoter is present. To test the role of hexonates in regulating expression of the hex genes, four distinct hexonate compounds were employed (D-galactonic acid; D-mannono-1,4-Lactone; L-(+)-gulonic acid γ-lactone and gluconate). All four could act as sole carbon and energy source for SE at 42 ˚C (hen body temperature). The hexonates induced distinct regulatory responses in the expression of the various hex genes, indicating that hex gene expression is controlled in response to hexonates, as expected, and that this response involves multiple regulatory pathways. However, the data are inconsistent with any role for hexonates in induction of hex genes by EW. EW, as expected, caused a major inhibition of SE growth, even when added at low levels (0.05%). In addition, the response of four hex genes (sen1436, sen1432, dgoR and sen2977) to EW was tested, and all four gave major induction effects (13-61 fold), confirming the previous report of EW induction of these genes. EW filtrate had little impact on EW-dependent hex gene induction, as did the provision of iron, temperature (30-42 ˚C) or pH (7-9). This finding indicated that an EW protein(s) of >10 kDa is responsible for the EW induction effect. Thus, four major EW proteins (albumin, conalbumin, ovomucoid and lysozyme) were tested for their ability to induce SEN1436 and a very strong induction effect (48 fold) was seen with lysozyme, suggesting this protein is primarily responsible for the EW-induction of the hex genes. Furthermore, three other lacZ fusions (SEN1432, dgoR and SEN2977) tested were also strongly induced by lysozyme (19-, 13- and 14-fold, respectively). This effect was confirmed with human lysozyme and with non-commercial sources of hen egg lysozyme. Thus, the results strongly suggest that lysozyme is the key factor in EW induction of hex gene expression; this is a novel finding. The SEN1432 and dgoR genes, encoding GntR-like regulators, were inactivated to determine their role in hex gene control. The deletions caused a moderate increase in the expression of the SEN1432- and SEN1436-, and dgoR-lacZ fusions, but no major effect on EW or lysozyme induction. Complementation largely reversed the expression effects of the mutations. Thus, the results indicate that neither DgoR nor SEN1432 are involved in the induction of the hex genes by EW lysozyme. The membrane-damaging antibiotic, polymyxin B (PMB), also caused a major induction of the hex genes, although not so great as that of lysozyme. Experiments with pmrA and phoP global-regulatory mutants showed that the PMB effect is controlled by the PhoPQ and PmrAB systems, but that the response to lysozyme is only slightly dependent on these regulators. This conclusion was supported by complementation with pmrAB. Thus, the control of the hex genes by the PmrAB and PhoPQ systems is complex, and involves additional factors. These results clearly show that the hex genes are subject to PMB induction and that this is largely controlled by PmrAB-PhoPQ. However, the response to lysozyme is only partly controlled by these factors indicating the involvement of another regulator. The results are consistent with a role for the observed hex gene induction by lysozyme in preserving the integrity of the cell envelope.