Positive Regulation of Staphylococcal Enterotoxin H by Rot (Repressor of Toxin) Protein and Its Importance in Clonal Complex 81 Subtype 1 Lineage-Related Food Poisoning

Abstract

Strains of this lineage produce staphylococcal enterotoxin H (SEH) in addition to SEA. However, an evaluation of the risk for the recently reported SEH has not been sufficiently conducted. We first searched for staphylococcal enterotoxin (SE) genes and SE proteins in milk samples that caused a large SFP outbreak in Japan. Only SEA and SEH were detected, while there were several SE genes detected in the samples. We next designed an experimental model using a meat product to assess the productivity of SEs and found that only SEA and SEH were detectably produced in situ. Therefore, we investigated the regulation of SEH production using a CC81 subtype 1 isolate. Through mutant analysis of global regulators, we found the repressor of toxin (Rot) functioned oppositely as a stimulator of SEH production. SEA production was not affected by Rot. seh mRNA expression correlated with rot both in media and on the meat product, and the Rot protein was shown to directly bind to the seh promoter. The seh promoter sequence was predicted to form a loop structure and to hide the RNA polymerase binding sequences. We propose Rot binds to the promoter sequence of seh and unfolds the secondary structure that may lead the RNA polymerase to bind the promoter, and then seh mRNA transcription begins. This alternative Rot regulation for SEH may contribute to sufficient toxin production by the CC81 subtype 1 lineage in foods to induce SFP. Staphylococcus aureus produces several virulence factors causing human and animal diseases. Among virulence factors produced by S. aureus, staphylococcal enterotoxins (SEs) show emetic and superantigen activities and are the causative agents for staphylococcal food poisoning (SFP) and toxic shock syndrome (1, 2). Presently, 23 SEs and SE-like toxins (SEls) have been reported. SEA-SEE are classical SEs, while SEG-SElX are newly described SEs/SEls (1, 2). Of these, SEA is shown to be the most important in SFP outbreaks (2). Conversely, reports suggest the newly described SEs also contribute to SFP outbreaks (3-5), but contributions for these SEs remain unknown. Thus, it is necessary to evaluate the importance of the newly described SEs in SFP outbreaks.Our previous report described the clonal complex 81 (CC81) subtype 1 lineage as the major SFP-associated lineage in Japan. Almost all of the CC81 subtype 1 isolates carried sea and showed high SEA production (6). Moreover, another unique genetic characteristic is the presence of seh. All CC81 subtype 1 isolates carried seh (all 30 strains), while other isolates rarely carried it (1 strain in 341 strains) (6).Though limited in numbers, previous epidemiological reports found SEH (seh) in SFP cases. In 2000, a large outbreak involving 13,420 patients consuming contaminated low-fat milk occurred in Japan (7,8). SEH and SEA were detected in the causative low-fat milk. A recent study in Germany showed almost all of the isolates from food poisoning by ice cream were positive for seh as well as sea (9). Further, an epidemiology study in South Korea showed that S. aure...

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