Virulence Factors Of Proteus Mirabilis Caused Diarrhea

Proteus mirabilis has been recognized as an opportunistic pathogen resulting in urinary tract infection, but its importance as a diarrhea pathogen is unclear. Proteus mirabilis produces several well characterized virulence determinants,however the association of the virulence factor to diarrhea has not been demonstrated.Foreign researchers generally believe that Proteus mirabilis isn’t the pathogen causing diarrhea and don’t list it in the category of food poisoning pathogen spectrum. At present, foreign scientists’research on Proteus mirabilis mainly concentrates on these strains causing urinary system infection and drug resistant. However no paper published and listed in Pubmed about the mechanism research of Proteus mirabilis causing diarrhea.We have isolated P.mirabilis as the sole microorganism from the outbreak cases of diarrhea and remaining food products; these isolates had the same pulsed-field gel electrophoresis (PFGE) pattern and could cause mice diarrhea or death which suggesting that these outbreaks were caused by P. mirabilis. Furthermore, the PFGE patterns of isolates recovered from outbreak patients differed from those recovered from the stools of healthy individuals, supporting the causality of diarrhea and indicating the potential for genome differences between strains. This has led us to hypothesize that some strains of P. mirabilis may contain virulence factors increasing their pathogenic and causing diarrhea.Methods and Results:1. Etiological and molecular characteristics of Proteus mirabilis that causing diarrheaTo analyze the etiological characteristics, virulence genes and plasmids carrying of diarrhea-causing Proteus mirabilis and to assess their relationship with drug resistance or pathogenicity.Proteus mirabilis, from six different sources (food poisoning, external environment and healthy people) were analyzed biochemically, on related susceptibility and pulsed-field gel electrophoresis (PFGE). Virulence genes were detected by PCR. Plasmids were exacted and sequenced after gel electrophoresis purification.The biochemical characteristics of Proteus mirabilis from different sources seemed basically the same, and each of them showed having common virulence genes, as ureC, rsmA, hpmA and zapA. However, the PFGE patterns and susceptibility of these strains are different, so as the plasmids that they carried. Plasmid that presented in the sequenced strain showed that the 2683bp length plasmid encodes qnrD gene was associated with quinolone resistance.Etiological characteristics and molecular characteristics of Proteus mirabilis gathered from different sources were analyzed. Results indicated that traditional biochemical analysis and common virulence gene identification might not be able to distinguish the strains with different sources. However, PFGE and plasmids analysis could distinguish the sources of strains and to identify shose plasmids that commonly carried by the drug-resistant strains. These findings also provided theoretical basis for further study on the nature of resistance and pathogenicity in Proteus mirabilis.2. Whole genome sequencing and comparison of Proteus mirabilisIn order to identify the specific genes in diarrhea causing strains,2 typical diarrhea causing strains (C05028 and C02011) were analyzed and compared with two control strains (1 from healthy people (B02005) and the other from environment sample (C02034)) via whole genome sequencing.Seven specific genes were found existed only in diarrhea causing strains. The function prediction shows that these genes encoding hemolysin, DNA nucleotide transferase, glycosyltransferases or have relationship with the transcriptional regulation of eukaryotic cells. These genes are discovered for the first time in the study.T4SS, the specific gene encoding type IV secretion system, is found in C02011 which isolated from food poisoning patient.3. The characteristic of type IV secretion system (T4SS) found in C02011The genomic comparison of this strain C02011 with the other P. mirabilis shows a recently inserted genomic island. An intact type IV secretion system (T4SS) is detected within this genomic island. Eleven T4SS genes are detected in the recently acquired genomic island, and their functions are annotated based on the NCBI blastp screening and protein structural characterization in the following sections. The major components of a T4SS are represented in P. mirabilis C02011 as lytic transglycosylases (VirB1), translocation energetics (VirB3-4, VirBl 1), mating channel (VirB2, VirB5-VirB10,lipoprotein), and cell attachment (VirB3-4), which suggests this T4SS is still functional after its re-location into P. mirabilis C02011.This T4SS has no nucleotide sequence similarity to any of the 8 currently avaiable P. mirabilis genomes, but has significantly homologs in two other gammaproteobacteria Klebsiella pneumoniae and Escherichia coli. The conserved topology of this T4SS further supports the recent horizontal transferring of the complete system into P. mirabilis C02011. So we may confidently claim that this genomic island is recently horizontally transferred from a microbe closely related to E. coli and K. pneumoniae. And this event occurs at least after the divergence of P. mirabilis C02011 from the other P. mirabilis strains.4. Isogenic deletion of virB9 gene in Proteus mirabilis strain C02011In order to determine the role of the virB9 gene in the pathogenesis of Proteus mirabilis, a virB9 isogenic in-frame deletion mutant was constructed.virB9 gene knockout strain is successfully constructed via using suicide plasmid PCVD442 and its intermediate host SM10 λpir. Moreover the revertant strain is also constructed for further study.Both C02011 and Pmil△virB9 have no significant difference of cell colony formation, growth ability and biochemical reaction in rich nutrient condition. Therefore the mutant strain is suitable for studying virulence infection of this gene.5. In vitro cell model for adhesion and invasion experiment was constructed.Human colon cancer cells (Lovo) and human colon adenocarcinoma cells (Caco-2) were selected for in vitro experiments to compare the invasion and adhesion level of Proteus mirabilis isolated from food poisoning patients and healthy human.The adhesion result showed that C02011 which isolated from food poisoning patients has strong adhesion ability on Lovo and Caco-2 cells. However the B02005 isolated from healthy people has weak invasion ability on Lovo and Caco-2 cell. The adhesion level of these two strains has a statistically significant difference (P<0.01). The invasion experiment showed the similar results. The Invasion level of these two strains has a statistically significant difference (P<0.01).Human colon cancer cell (Lovo) is more sensitive and suitable for the adhesive and invasive ability study of Proteus mirabilis.6. The virB9 deletion led to a significant reduction of adhesion and invasion ability to Lovo cell for C02011The in vitro adhesion results showed that the adhesion rate of P13006 was significantly lower than that of their parent strain C02011 (P<0.001). Similarly, the virB9 deletion led to a significant reduction of invasion ability for C02011 (P<0.001).7. An animal model of diarrhea was constructed.To test the virulence of Proteus mirabilis strains causing diarrhea and construct an animal model,6 Proteus mirabilis strains were used to infect mice. No mice dead by intragastric administration of any strain, but these strains isolated from food poisoning samples can cause the change of mice feces shape. All mice died 7 hours later after intraperitoneal injection of strains isolated from diarrhea patient.The feces of mice were soft and pathological change was observed in mice intestinal. However the mouse injected with strains isolated from healthy human and external environment samples have normal vitality and morphology.The mice diarrhea model of Proteus mirabilis wad successfully established. The virulence of strains isolated from food poisoning samples is obviously stronger than that from healthy human and environmental. The Proteus mirabilis isolates from food poisoning samples can cause mice diarrhea and even death, including pathological histological change.8. The deletion of virB9 can reduce the pathogenicity of Proteus mirabilis C02011 on mice.To identify the function of virB9 gene, in vivo study was carried on the animal model to observe diarrhea symptom and pathological change of infected animals. The virulence of C02011 wild strains was stronger than that of virB9 gene knockout strains, which can cause diarrhea, water content of intestinal increasing and the intestinal pathological change. VirB9 knockout strains failed to induce mice diarrhea and water content of intestinal had no statistical difference from the saline control. However the intestinal pathological slice of showed that there was still a certain inflammatory response existed, which means that virB9 gene is not the unique virulence gene in C02011. The virB9 knockout strain still has the invasion ability of mice intestinal tissue. Further experiments needed for verification.9. Multiple real-time PCR assay was developed to detect Proteus mirabilis causing diarrhea.Based on Homo-Tag Assisted Non-Dimer system and modified molecular beacon probe, multiple real-time PCR assay was developed to detect the specific virulence genes of Proteus mirabilis causing diarrhea, which newly discovered in the study.The method can be widely used for rapid detection of food poisoning or food contaminants, distinguish normal strains from diarrhea causing strains.Statistical analysisAll values are expressed as mean ± standard deviation. Statistical analysis was performed with Student’s t-test for comparison of two groups, and with ANOVA for multiple comparisons. In vivo experiments data were analyzed by Pearson Chi-Square test. Differences with P<0.05 were considered to be statistically significant.ConclusionAfter whole genome sequencing and comparison of Proteus mirabilis, which isolated from diarrhea patient and healthy human, some specific virulence genes are found only in diarrhea strains. The most unique discovery is the existence of T4SS system in the C02011 isolated from diarrhea patients’ vomit. This T4SS has no nucleotide sequence similarity to any of the 8 currently avaiable P. mirabilis genomes, but has significantly homologs in two other gammaproteobacteria Klebsiella pneumoniae and Escherichia coli. The conserved topology of this T4SS further supports the recent horizontal transferring of the complete system into P. mirabilis C02011. So we may confidently claim that this genomic island is recently horizontally transferred from a microbe closely related to E. coli and K. pneumoniae. And this event occurs at least after the divergence of P. mirabilis C02011 from the other P. mirabilis strains.The virB9 deletion led to a significant reduction of adhesion and invasion ability to Lovo cell for C02011,so as its pathogenicity on mice(P<0.01). T4SS system is a crucial pathogenic factor of Proteus mirabilis C02011, which might be a key factor causing diarrhea and vomiting. The function of the other genes in T4SS system will be the key research fields in the follow-up study.