Molecular Dissection Of The Pathogenesis Of Highly Pathogenic Infections By Streptococcus Suis Serotype 2

Streptococcus suis serotype 2 (SS2) is an important zoonotic pathogen associated with a wide range of diseases in pigs, including meningitis, septicaemia, pneumonia, endocarditis, arthritis and sudden death, and has spread over 20 countries, resulting in great economic losses in pig-industries each year. Occasionally, SS2 can also be transmitted to human beings through wound contamination, causing sporadic cases of human with the hallmarks of meningitis, septicemia, arthritis, etc, and it is responsible for more than 200 cases of severe infections in humans worldwide since its first discovery in Denmark in 1968. However, two major emerging infectious disease outbreaks of SS2 in China (one in Jiangsu Province, 1998, and the other in Sichuan Province, 2005), raised considerable international concerns among the public health professionals. A key feature of these two Chinese outbreaks is the prevalence of streptococcal toxic shock syndrome (STSS) manifesting itself as acute high fever, vascular collapse, hypotension, shock, multiple organ failures, short course of disease and high lethality(81.3%~97.4%).To elucidate the high pathogenicity of SS2 isolates in China, we isolated two SS2 strains, namely 05JYS68 and 07NJH06, from tonsil sample of healthy pig and CSF sample of encephalomeningitis patient from prevalent regions of Jiangsu province, respectively. The bacterial isolate was examined using microscopy observation, slide agglutination test, multiplex-PCR assay, as well as animal infection experiments. Multiple lines of evidence identified both the isolates as SS2. PCR test evidences indicated that several putative virulence-factor genes (mrp, epf and suilysin) is absent in 05JYS68 strain, and infection experiments indicated that 05JYS68 strain is avirulent in swine model of infection. The 07NJH06 strain exhibited the most putative virulence-factor genes including gdh, mrp, epf, suilysin, fbp, srtA and cps2J. PCR evidences indicated that the pathogenicity island PAI89K was absent in the genome of the 07NJH06 strain, however, two disjunction genes(05SSU0942 and 05SSU0965)within PAI89K were detected. Furthermore, the 07NJH06 strain was virulent in murine and swine model of infection, even its virulence was lower than the endemic strain 05ZYH33. The identification of 05JYS68 and 07NJH06 will facilitate to understand the pathogenicity of SS2 and development of effective strategies to combat highly pathogenic SS2 infections.To shed light on the mystery of high virulence of the epidemic outbreak strains of SS2, whole genome sequencing and comparative genomics analysis of 3 different Chinese SS2 strains (98HAH12, 05ZYH33 and 05JYS68) were undertaken. According to genome phylogenetic analysis, Chinese SS2 isolates can be divided into two different phylogenetic species. In genome of 05JYS68, 35 Large-scale arrangements affect little of the gene content, and each of the strains gained or lost some special function genes for its virulence and/or environment adaptive respectively. Interestingly, a candidate pathogenicity island (PAI) of ~89kb in length, which is designated 89K and specific for Chinese SS2 virulent isolates, was unveiled at the genomic level. It shares the universal properties of PAIs such as distinct GC content, consistent with its pivotal role in STSS and high virulence. To our knowledge, this is the first PAI candidate from S. suis worldwide. Our finding thus sheds light on STSS triggered by SS2 at the genomic level, facilitates further understanding of its pathogenesis and points to directions of development on some effective strategies to combat highly pathogenic SS2 infections.Sortase A (SrtA), originally identified as a transpeptidase in Staphylococcus aureus, plays key roles in full virulence of pathogenic bacteria. In silico search in the genome of a Chinese virulent SS2 strain, 05ZYH33, revealed six putative genes encoding sortases or sortase-like proteins together with thirty-three genes corresponding to surface proteins with the motif of LPXTG. On the basis of the criterion proposed by Dramsi et al., SrtA of 05ZYH33 was considered to be a class A enzyme and the other five sortase homologs to be class C subfamily 3 enzymes. Notably, two putative pilus gene clusters, designated srtBCD and srtF, were identified in 05ZYH33, and similar genetic organization phenomena have been observed in pilus-associated gene clusters of some gram-positive bacteria. Given its potential role of srtA in bacterial pathogenesis, it is of the utmost interest to examine whether incorrect surface presentation due to srtA inactivation would exert a general impact on the virulence of Chinese epidemic SS2 strain as it appears to in the other pathogens (Listeria monocytogenes, S. mutans, S. agalactiaeand S. pneumonia). An isogenic srtA mutant (ΔsrtA) of 05ZYH33 strain was obtained by homologous recombination. Immunofluorescence analysis revealed that two known virulence-associated surface proteins featuring LPXTG motif, muramidase-released protein (MRP) and surface antigen one (Sao), were absent in theΔsrtA. Piglet infection experiments showed that deletion of srtA attenuated the full virulence of 05ZYH33 strain, and impaired its colonizing potential in specific organs. Furthermore, theΔsrtA displayed significant reduction in adherence to human cells (Hep-2 and HUVEC). Collectively, we concluded that SrtA was involved in the virulence manifestation of STSS-causing Streptococcus suis serotype 2.