Functional Analysis Of The Specific Genes In Salmonella Serogroup C1

Salmonella is the most common foodborne pathogen, which often tops the list in bacterial food poisoning worldwide. Based on differences in the antigenic structure, the Salmonella genus is diverse with over 2600 serovars, which are classified into 46 serogroups. Nearly all humans-infected pathogenic strains belong to serogroups A, B, C1, C2 and D. Many highly virulent serovars(e.g. S. Choleraesuis, S. Infantis, and S. Paratyphoid C) are from Salmonella serogroup C1, however, this serogroup has not received sufficient attention from researchers, specifically the synthesis of their unique O-antigen. Relying on the platforms of Salmonella genomics and bioinformatics, we recently found seven genes that were conserved and specific for Salmonella serogroup C1 by comparative genomic approach. According to preliminary analyses, most of these C1-specific genes encode membrane proteins with high numbers of transmembrane segments(TMs), such as SC2092, SC0368 and SC0595. Their functional analysis and verification will provide experimental evidence to further understand the correlation between serotype and genotype.In this study, we firstly predicted the structures and functions of these C1-specific genes and their encoded proteins using bioinformatics methods. And then, after constructing the in-frame deletion mutants by homologous recombination, we further demonstrated that these genes’ deletion affected Salmonella serum agglutination, growth state, cell structure, motility and so on. These findings will allow a better understanding of the role of these specific genes in Salmonella serogroup C1, and help us to evaluate their effects on bacterial structure and physiology. Subsequently, the key genes and regulatory pathways affected by C1 serogroup-specific genes were revealed at the transcriptional level by RT-q PCR and/or RNAseq. Finally, we obtained the functions of the C1-specific genes and influences on biological characteristics of serogroup C1 strains(with S. Choleraesuis ATCC 10708 as a representative strain). The main contents and results are as follows:1)Functional prediction of C1-specific genes by bioinformatics methods The nucleotide and the corresponding amino acid sequences of serogroup C1-specific genes(SC2092, SC0368 and SC0595) were respectively download from the NCBI website(http://ncbi.nlm.nih.gov/). Blastn(p) software was used for their homologous comparison, while TOPCONS software was used for topology prediction of these membrane proteins. The results showed that: a) These genes and their encoded proteins are highly conserved and specific in Salmonella serogroup C1, which means they are present in all the strains belonging to C1-group, but absent in other Salmonella serogroups or non-Salmonella strains; b) C1-specific gene SC2092 putatively encodes a protein with 12 TMs is within the O7 antigen rfb gene cluster, which is one of the important features of O antigen flippase(Wzx). Although gene SC2092 and its deduced amino acid sequence share little sequence similarity with the wzx genes/Wzx proteins of S. Typhimurium(0.8% and 23%, respectively) and E. coli O157:H7(4% and 24%, respectively), the topology prediction profile of the putative Wzx ortholog(the SC2092 gene product) from S. Choleraesuis was highly similar to Wzx proteins from S. Typhimurium and E. coli O157:H7. All of the programs predicted that the N and C termini of the three above-mentioned proteins were located in the cytoplasm. The number of TMs and the orientation of the intervening loops for the three Wzx proteins predicted by different algorithms gave similar results; c) C1-specific gene SC0368 and SC0595 putatively encoded identical topology prediction structure proteins with 10 TMs is outside of the O7 antigen rfb gene cluster. The two genes and their deduced amino acid sequences have a high degree of sequence homology, 87% for nucleotide and 83% for amino acid.2)Vector construction for homologous recombination and screening of inframe deletion mutants Primers were designed according to the principle of nested PCR, and the fragment of homologous arms were amplified by two rounds of PCR. The fragment of homologous arms were ligated into suicide vector p RE112, which carries a chloramphenicol resistance gene and a sucrose-sensitivity gene sac B. The resulting plasmid p RE△wzx, p RE△0368 and p RE△0595 were transformed by electroporation(2500 V, 5 ms) into E. coli SM10λpir. The marker-free in-frame deletion mutants of △wzx C1, △0368, △0595 and △0368△0595 were screened by double selection in two steps: co-conjugation and sugar screening. These mutants provide experimental materials for further comparison of biological characteristics and high-throughput RNA sequencing.3)Correlation analysis between serogroup C1-specific genes and the synthesis of bacterial surface antigens Slide agglutination test and SDS-PAGE analysis of lipopolysaccharide(LPS) were used to analyze the changes in surface antigens of these deletion mutants. The results were shown as follow: a) SDS-PAGE revealed that LPS biosynthesis, specifically O-antigen synthesis, was incomplete in an SC2092 in-frame deletion mutant, and no agglutination reaction with the O7 antibody was exhibited in this mutant. Therefore, it was revealed that this Salmonella serogroup-C1-specific gene SC2092 encoded a putative flippase, which was required for O7-polysaccharide biosynthesis, and was designated here as wzx C1. b) Positive agglutination reactions were obtained in SC0368 in-frame deletion mutant △0368, SC0595 in-frame deletion mutant △0595, and the double mutant △0368△0595. Compared with the wild type, no significant visible differences was found in the three mutants, indicating that the two genes(SC0368 and SC0595) are not related to the synthesis of O7 antigen. However, SC0368 gene deletion affected the synthesis of HC antigen.4)Phenotypic characteristics of deletion mutants in serogroup C1-specific genes a) The wzx C1 mutant lacked swarming motility on solid surfaces and was impaired in swimming motility in soft agar. Moreover, microscopic examination and RT-q PCR exhibited that an increased auto-aggregation and a strong defect in flagella expression, respectively, were responsible for the reduced motility in this mutant. In addition, the wzx C1 mutant was more sensitive than the wild-type strain to Na Cl, and auto-aggregation of mutant cells was observed immediately up on the addition of 1% Na Cl to the medium. Interestingly, the motility deficiency of the △wzx C1 mutant strain, as well as the cell agglomeration and the decrease in flagellar expression, were relieved in a Na Cl-free medium. In all, our results show that the mutation of wzx C1 triggers a Na Cl-dependent motility deficiency. b) The SC0368 mutant significantly increased the bacterial growth rate in the stationary phase. The growth population of △0368 and △0368△0595 mutants(ca. 1010 CFU/m L) was two magnitude higher than that of the wild-type and △0595 mutant(ca. 108 CFU/m L). The results of flagella silver staining, microscopic examination and motility assays showed that the flagella cannot be synthesized and motility defects in these SC0368 gene deletion mutant strains(△0368 and △0368△0595). c) Compared with the wild-type, no significant effect on the biological characteristics was found in SC0595 gene deletion mutant.5)Analysis and verification of differently expressed genes by RNAseq and RT-q PCR methods For a more comprehensive understanding of the relationship between SC0368 and SC0595 and their effects on bacteria, RNAseq, a high-throughput transcriptional technology, was used to analyze differently expressed genes in △0368, △0595 and △0368△0595 mutants, relative to wild-type. And RT-q PCR methods was used to verify key genes. The results were shown as follows: a) C1-specific genes SC0368 and SC0595 have synergistic effect on the synthesis of LPS. Compared with the wild-type, 6 genes associated with LPS core synthesis were significantly reduced only in △0368△0595 mutant. b) In △0368 and △0368△0595 mutants, 20 genes from two pathways related with flagella synthesis and chemotaxis appeared significantly lower expression. These differently expressed genes were belonging to flagellar regulon class 3, responsible for filament assembly, flagellar rotation, and chemotaxis. In which, the gene fli A(encoded flagellar sigma factor σ28), which directs transcription to class 3 promoters, might be one of the key factors firstly affected by SC0368 directly or indirectly.In summary, bioinformatics prediction, phenotypes comparative analysis of deletion mutants, and transcriptional level(RNAseq and RT-q PCR) evaluation, were used to explore the function and the influence of Salmonella serogroup C1-specific genes SC2092, SC0368 and SC0595. This is the first study to experimentally demonstrate a connection between Salmonella serogroup specific genes identified by comparative genomics with the synthesis of specific O-antigen biosynthesis or/and LPS composition. Our data of phenotype characteristics and transcriptional analysis will enrich bacterial molecular regulatory mechanisms on growth, motility, adhesion and so on. These results will provide a better understanding of Salmonella serogroup C1.