Differential gene expression profile of Shigella dysenteriae causing bacteremia in an immunocompromised individual

Aim: Shigella species has varying levels of virulence gene expression with respect to different sites of infection. In this study, the differential gene expression of S. dysenteriae in response to its site of infection was analyzed by transcriptomics. Methods: This study includes four clinical Shigella isolates. Transcriptomics was done for the stool and blood samples of a single patient. Isolates were screened for the presence of antimicrobial resistance genes. Results: The majority of genes involved in invasion were highly expressed in the strain isolated from the primary site of infection. Additionally, antimicrobial resistance (dhfr1A, sulII, blaOXA. blaCTX-M-1 and qnrS) genes were identified. Conclusion: This study provides a concise view of the transcriptional expression of clinical strains and provides a basis for future functional studies on Shigella spp.

Diarrheal disease is the second leading cause of mortality in children according to WHO [1]. Shigella spp. is one of the important causes of dysentery globally and causes severe and occasionally life-threatening diarrheal infection. In Asia, it is estimated that there are 125 million infections and 14,000 deaths due to shigellosis annually [2]. Clinically, the infection may lead to rare but potentially fatal extra-intestinal complications like bacteremia. Though, bacteremia due to Shigella spp. is rare, it is reported in 0.4-7% of the cases. Notably, young age, malnutrition and immunosuppression are known to be the risk factors for Shigella spp. bacteremia [3].
Bacteria have developed various mechanisms to adhere to the organ surfaces. Some bacteria can adopt an intracellular lifestyle and get internalized inside various host cell types to replicate. Finally, pathogenic bacteria can get access to deeper tissues using various mechanisms to cross mucosal barriers and access the bloodstream, which is a gateway for all host organs [4].
Pathogens showing a variable expression of virulence factors have been observed. In fact, the expression of virulence factors depends largely on the environmental conditions. This expression of virulence genes is induced under conditions similar to those found at the site of invasion. Studies have demonstrated that a temperature of 37 • C is a favorable growth condition for bacteria in intestinal epithelial cells, but bacteria grown at 30 • C can be phenotypically avirulent and noninvasive [5]. The bacterium can be found either in the intestinal lumen, inside epithelial cells, phagocytes or in the bloodstream. The expression level of virulence factors in these different locations varies accordingly in order to counteract different host defense mechanisms, as reported earlier by Ribet and Cossart [4]. In this study, Shigella strains causing bacteremia were characterized using RNA-Sequencing to identify genes that are differentially expressed based on the site of infection. The genes responsible for invasion, virulence, stress, antimicrobial resistance (AMR) and other genes involved in cellular metabolism are also discussed.

Strains
This study reports four cases of Shigella bacteremia diagnosed between the years 2015 and 2018. The identified isolates include two isolates each of S. flexneri serotype 2 and S. dysenteriae serotype 9. The isolates were confirmed by standard biochemical tests [6]. The isolate was serotyped using commercial antisera as per the manufacturer's instructions (Denka Seiken, Tokyo, Japan). For transcriptome analysis, stool (FC3355) and blood (BA42767) samples of the sole patient were studied further. Patient's symptoms, clinical diagnosis and outcome were detailed in Table 1. The term invasive (sterile site) and noninvasive (nonsterile site) refer to the pathogen isolation site in this study.

RNA isolation
Total RNA was extracted using RNeasy Mini kit (Cat#74106, Qiagen, GmBH, Germany) according to the manufacturer's recommendations. The RNA was checked using the Qubit R 3.0 Fluorometric Quantitation kit (Invitrogen, Merelbeke, Belgium).

RNA-sequencing & analysis
The invasive traits of selected isolates were studied by comparing the differential gene expression profile of the strain isolated exclusively from stool and blood specimen concurrently by transcriptomics. RNA-sequencing procedure was performed according to the manufacturer's instructions using Ion Torrent (PGM) sequencer with 400-bp read chemistry (Life Technologies, CA, USA) [10]. The quality and quantity of each library was determined at each step with a Qubit R 3.0 Fluorometer. De novo assembly using AssemblerSPAdes and annotation through RNA-Seq analysis was performed in PATRIC, the bacterial bioinformatics database and analysis resource.

Statistical analysis
In this study, greater than twofold changes in the gene expression level between two variables was considered significant. Results were analyzed for correlation and tested for significance by Student's t-test (p < 0.05). SPSS 16.0 and Microsoft Excel 2007 (IL, USA) were used for the statistical evaluation.

Results & discussion
Shigella infection is in the majority confined to the GI tract which invades the colonic mucosa but rarely penetrates further into deeper tissues [11]. This study discusses four cases of Shigella bacteremia. The risk factors observed in these patients were diabetes, malignancy and immunosuppressant therapy. Previous literatures on mechanisms of pathogenesis have been described for S. flexneri. However, the present study has shown the invasion process of S. dysenteriae serotype 9.
AMR gene PCR Among the four isolates studied, only two isolates harbored AMR genes that codes for β-lactams, trimethoprim/sulfamethoxazole, fluroquinolones and cephalosporins, whereas no AMR genes were identified in the other two isolates. The resistance genes obtained in this study were found to be a common profile seen in the genus. The results were given in Table 2. AMR was generally more common in Shigella than in other enteric bacteria [12].
Differential gene expression analysis S. dysenteriae serotype 9 obtained from stool and blood specimen of the single patient was studied. In RNA-Seq analysis, significant fold change was observed between non-invasive Sd FC3355 and invasive Sd BA42767 strains for the genes involved in invasion, virulence, motility and other cellular processes. Totally 56 genes were differentially expressed between the strains. Of these, few genes were expressed only in invasive strain Sd BA42767 like csp, dcm, hisE and enterotoxin genes with reduced expression, this showed the significance of these genes in the invasive phenotype of the strain. The majority of the genes (44/56 genes) were highly expressed in non-invasive isolate from the gut, which is the primary site of invasion for Shigella infection. Genes with no expression data were excluded from the analysis. The genes analysed were given as a supplementary material.

Motility-associated genes
Shigella pathogenesis involves bacterial invasion and spread through colonic mucosa [13]. Shigella spp. are able to move through the cytoplasm of host cells and into adjacent cells by polymerizing actin [14] which is mediated by IcsA (virG), encoded on the 220-kb virulence plasmid [15,16]. We observed that IcsA protein was expressed only in noninvasive Shigella isolate (Table 3). This correlates with the fact that IcsA is required for inter-and intracellular spreading of Shigella within the host intestinal epithelium. VirK gene, which is required for post-transcriptional regulation of icsA expression, has also been expressed.

Virulence/invasion associated genes
Shigella virulence plasmid is an essential virulence determinant of the species and encodes the molecular machinery necessary for tissue invasion and intracellular survival. The virulence plasmid encodes the 30 kb Mxi-Spa type III secretion system (T3SS) and invasion plasmid antigens (Ipa proteins) required for invasion of the colonic and rectal   epithelial cells and cell-to-cell spread of the bacteria, resulting in the symptoms of bacillary dysentery [17,18]. Shigella pathogenesis mainly relies on the Mxi-Spa T3SS and its effector proteins [19]. The invasion plasmid antigen (ipaH) gene, which was reported to be carried by all four Shigella species, was found to be highly expressed in invasive isolate in this study, whereas ipaD, a host injection protein was expressed only in noninvasive isolate. Further, ipgA, B, C, D, F known to facilitate local invasion in to epithelial cells, were also expressed only in noninvasive isolates (Table 3). Therefore, the virulence plasmid is the key molecular signature of Shigella spp. pathogenesis and is fundamental for initiating infection and manipulating the immune response of the host [18]. PhoQ/PhoP is a two-component system that governs virulence and regulates several cellular activities in Shigella spp. [20]. In the present study, PhoP was highly expressed in invasive isolate, whereas PhoQ showed no significant difference in the expression level. In addition, BarA-UvrY two-component system was shown to have increased expression in invasive isolate. This system also controls the activity of CsrA (carbon storage regulator) protein which regulates carbon metabolism, flagellar biosynthesis and biofilm formation. This process has been previously reported in uropathogenic E. coli [21]. We observed that CsrA protein was upregulated in noninvasive isolate.

Stress-associated genes
Bacteria have developed a number of mechanisms to adapt the changing environmental conditions within the cells. One such mechanism is the production of small cold shock proteins (Csp) to counteract the sudden temperature downshift. Csps have been shown to contribute to osmotic, oxidative, starvation, pH and ethanol stress tolerance as well as to host cell invasion [22]. CspA is a major cold shock protein, first described in E. coli [22] was found to have significant differences in the expression level between the invasive and noninvasive isolate. Similarly, CspD and CspE proteins showed significant differences in their expressions, whereas CspC was highly and solely expressed in invasive isolate (Table 3). Another defense mechanism against various environmental stresses is the production of heat shock proteins. Heat shock proteins that are important for cell survival and are usually related to the virulence of the pathogens have been expressed in both the isolates [23].

Genes involved in metabolism
In this study, ilv proteins such as ilvA, B, C, D, E and N involved in amino acid biosynthesis showed significantly increased expression in invasive isolate. Histidine (his) proteins like hisA, B, C, D, E, F, G and H were found to have significant upregulation in invasive isolate. Further, member of the two-component regulatory system NtrB/NtrC and other regulator proteins like NarL and NarX involved in the regulation of nitrogen was expressed in both the isolates with no significant difference in the expression level. Similarly, several other genes such as (ygiX, qseC, rcsC, rcsB, rstA, rstB, yfhA, yfhK, dcuA, baeR, baeS, vsr) were present but showed no significant difference between the isolates.

Cellular process & signaling
During Shigella infection, certain effector proteins promote cell survival. IpgD which associated with increased intracellular bacterial replication [24] was highly and solely expressed in noninvasive isolate as expected. Further ospC and virA were also found to be expressed in noninvasive isolate [24]. DNA methylation is an important component in numerous cellular processes and plays an important role in regulating gene expression [25,26]. DNA cytosine methyltransferase protein was only slightly expressed in invasive isolate in this study.

Uncharacterized genes
Two genes encoding uncharacterized proteins were identified. Uncharacterized innermembrane transporter YedA gene was not expressed in the study isolates, which has been previously identified as hypothetical protein in S. dysenteriae strain Sd197. Another gene named YobF, which is a small protein with no known function showed significantly increased expression in invasive isolate. Yet the functions of these genes remain obscure.

Conclusion & future perspective
Shigella spp. is a highly contagious pathogen and humans are the only reservoir that spreads through fecal-oral contamination. The invasive ability of this pathogen is a key determinant in the establishment of the disease. The invasive phenotype of Shigella spp. is linked to the expression of various effector/regulatory genes. The differential protein expression by S. dysenteriae serotype 9 observed in this study suggests that it has a specific response to particular intracellular environment. Notably, many uncharacterized genes with unknown functions demonstrate the complexity of the regulatory network in S. dysenteriae. These genes needs to be further characterized to understand unidentified strategies for infection and successful survival of this pathogen. Further, the in vivo mechanism of S. dysenteriae invasion are difficult to fully study until the intracellular environment is mimicked in vitro. To the best of our knowledge, this is the first Indian study that compares the gene expression profile of clinical S. dysenteriae serotype 9 with respect to their invasion.

Executive summary
• Most of the earlier studies on mechanisms underlying pathogenesis was derived from Shigella flexneri. However, the present study shows the invasion process of Shigella dysenteriae serotype 9. • RNA sequencing was done to study the differential expression of genes involved in the invasion process of the pathogen with the respect to the infection site. • On virulence analysis, enterotoxin gene (set) and invasion associated genes such as ipaH and ial was identified in two, one and three isolates, respectively. • For antimicrobial resistance, only two isolates harbored genes that codes for β-lactams, trimethoprim/sulfamethoxazole, fluroquinolones and cephalosporins resistance. • RNA-Seq analysis showed significant fold change between noninvasive Sd FC3355 and invasive Sd BA42767 strains for the genes involved in invasion, virulence, motility and other cellular processes. • Majority of the genes (44/56 genes) were highly expressed in noninvasive isolate, which is the primary site of invasion for Shigella spp. Few genes were expressed only in invasive isolate Sd BA42767, which shows the significance of these genes in the invasive phenotype of the strain. • This study explores that Shigella spp. has a specific response to particular intracellular environment. future science group www.future-science.com