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Alteration in apyrase enzyme attenuated virulence of Shigella flexneri

BangaSingh KK, Nisha M, Lau HY, Ravichandran M, Salleh MZ

Microb Pathog, 2016 Feb;91:123-8.
PMID: 26706344 DOI: 10.1016/j.micpath.2015.12.004

Virulence of Shigella is attributed to the genes presence in chromosome or in the megaplasmid. The apy gene which is located in the megaplasmid of Shigella species encodes for apyrase enzyme, a pathogenesis-associated enzyme causing mitochondrial damage and host cell death. In this study we constructed an apy mutant of Shigella flexneri by insertional activation using a kanamycin resistant gene cassette. The wild type apy gene of S. flexneri 2a was PCR amplified, cloned and mutated with insertion of kanamycin resistant gene cassette (aphA). The mutated construct (apy: aphA) was subcloned into a conjugative suicidal vector (pWM91) at the unique Sma1 and Sac1 sites. The mutation of the wild apy gene in the construct was confirmed by DNA sequencing. The mutated construct was introduced into wild type S. flexneri 2a by conjugation with Escherichia coli. After undergoing homologous recombination, the wild apy gene was deleted from the construct using the sucrose selection method. Non-functional activity of the apyrase enzyme in the constructed strain by colorimetric test indicated the successful mutation of the apyrase enzyme. This strain with mutated apy gene was evaluated for its protective efficacy using the guinea pig keratoconjunctivitis model. The strain was Sereny negative and it elicited a significant protection following challenge with wild S. flexneri strain. This apy mutant strain will form a base for the development of a vaccine target for shigellosis.

Matched MeSH terms: Shigella flexneri/enzymology*; Shigella flexneri/genetics; Shigella flexneri/pathogenicity*
Fulltext Temperature-regulated expression of outer membrane proteins in Shigella flexneri

Harikrishnan H, Ismail A, Banga Singh KK

Gut Pathog, 2013;5(1):38.
PMID: 24330657 DOI: 10.1186/1757-4749-5-38

Bacteria exist widely in a diversity of natural environments. In order to survive adverse conditions such as nutrient depletion, biochemical and biological disturbances, and high temperature, bacteria have developed a wide variety of coping mechanisms. Temperature is one of the most important factors that can enhance the expression of microbial proteins. This study was conducted to investigate how outer membrane proteins (OMPs) of the bacterium Shigella flexneri respond to stress, especially during fever when the host's body temperature is elevated.

Matched MeSH terms: Shigella flexneri
Fulltext Shigella vulvovaginitis in a three-year-old child: a case report

Cheong YM, Jegathesan M, Lim YS

Med J Malaysia, 1984 Mar;39(1):92-4.
PMID: 6392838

This is a report of a case of vulvovaginitis due to Shigella flexneri in a three-year-old child. This is probably the first documented case of shigella vulvovaginitis In Malaysia. The patient was successfully treated with cotrimoxazole. Extraintestinal infections by Shigella are rare and are briefly reviewed in this article.

Matched MeSH terms: Shigella flexneri/isolation & purification
Fulltext Shigella flexneri serotype 1c derived from serotype 1a by acquisition of gtrIC gene cluster via a bacteriophage

Tang SS, Carlin NI, Talukder KA, Cam PD, Verma NK

BMC Microbiol, 2016 Jun 27;16(1):127.
PMID: 27349637 DOI: 10.1186/s12866-016-0746-z

BACKGROUND: Shigella spp. are the primary causative agents of bacillary dysentery. Since its emergence in the late 1980s, the S. flexneri serotype 1c remains poorly understood, particularly with regard to its origin and genetic evolution. This article provides a molecular insight into this novel serotype and the gtrIC gene cluster that determines its unique immune recognition.
RESULTS: A PCR of the gtrIC cluster showed that serotype 1c isolates from different geographical origins were genetically conserved. An analysis of sequences flanking the gtrIC cluster revealed remnants of a prophage genome, in particular integrase and tRNA(Pro) genes. Meanwhile, Southern blot analyses on serotype 1c, 1a and 1b strains indicated that all the tested serotype 1c strains may have had a common origin that has since remained distinct from the closely related 1a and 1b serotypes. The identification of prophage genes upstream of the gtrIC cluster is consistent with the notion of bacteriophage-mediated integration of the gtrIC cluster into a pre-existing serotype.
CONCLUSIONS: This is the first study to show that serotype 1c isolates from different geographical origins share an identical pattern of genetic arrangement, suggesting that serotype 1c strains may have originated from a single parental strain. Analysis of the sequence around the gtrIC cluster revealed a new site for the integration of the serotype converting phages of S. flexneri. Understanding the origin of new pathogenic serotypes and the molecular basis of serotype conversion in S. flexneri would provide information for developing cross-reactive Shigella vaccines.

Matched MeSH terms: Shigella flexneri/genetics*; Shigella flexneri/immunology; Shigella flexneri/virology*
A 35 kDa antigenic protein from Shigella flexneri: in silico structural and functional studies

Yung-Hung RL, Ismail A, Lim TS, Choong YS

Biochem Biophys Res Commun, 2011 Nov 18;415(2):229-34.
PMID: 21982766 DOI: 10.1016/j.bbrc.2011.09.116

Shigella flexneri serotype 2a is a major public health concern in the developing and under-developed countries which contributes to shigellosis endemic and mortality. Thus, there is an urgent need for a rapid diagnostic test for effective therapy and disease management. Previous study showed that a ∼35 kDa antigenic protein from S. flexneri is a potential biomarker. We therefore modelled the three-dimensional structure of the antigen to probe its functionality which could aid in the development of an antigen-based diagnostic. Results showed that the antigen is a transmembrane protein consists of OmpA and OmpA-like domains. The OmpA domain is a beta-barrel embedded in the outer membrane with four surface-exposed extracellular loops. The OmpA-like domain is linked to the OmpA domain with a 17 amino acids linker and located in the periplasmic. Docking of peptidoglycan into the groove of OmpA-like domain might help in catalyzing the bacterial cell wall formation. Both domains are expected to be involved in the virulence, structural stability, pathogenesis and survival of Shigella thus made the 35 kDa protein a suitable shigellosis diagnostic biomarker. This structural elucidation will also enable a better identification of the epitope regions for the development of specific binders to the 35 kDa antigen.

Matched MeSH terms: Shigella flexneri/immunology*
Fulltext Fulminant hepatic failure and shigella bacteremia

Abidin Z, Iyngkaran N, Puthucheary SD

Med J Malaysia, 1983 Jun;38(2):112-3.
PMID: 6353185

A three and a half year old boy with shigellosis developed fulminant hepatic failure. The hepatic derangements rapidly improved over a period of two weeks after treatment of the shigellosis with parenteral gentamicin. We believe this is the first documented report of fulminant hepatic failure due to shigella sepsis.

Matched MeSH terms: Shigella flexneri/isolation & purification
Fulltext Bacterial pathogens from the intestinal tracts of various species of cockroaches

Rampal L, Oothuman P, Jeffery J, Daud MZ, Shekhar C, Senan P, et al.

Med J Malaysia, 1983 Jun;38(2):104-7.
PMID: 6353184

Bacterial isolates were made from the intestinal tracts ofcarious species of cockroaches (Periplaneta americana, Periplaneta brunnea, Periplaneta australasiae, Neostylopyga rhombifolia, Nauphoeta cinerea) trapped from kitchens and stores (houses and hospital), Shigello, flexneri, Salmonella typhi, Escherichia coli and Salmonella sp. were some of the bacteria isolated and identified.

Matched MeSH terms: Shigella flexneri/isolation & purification
Ultrasensitive pathogen detection with a rolling circle amplification-empowered multiplex electrochemical DNA sensor

Yeap CSY, Chaibun T, Lee SY, Zhao B, Jan Y, La-O-Vorakiat C, et al.

Chem Commun (Camb), 2021 Nov 16;57(91):12155-12158.
PMID: 34726213 DOI: 10.1039/d1cc05181d

We report a highly sensitive and selective multiplex assay by empowering an electrochemical DNA sensor with isothermal rolling circle amplification. The assay could simultaneously detect and discriminate three common entero-pathogens in a single reaction, with femtomolar sensitivity. It is useful for field- or resource-limited settings.

Matched MeSH terms: Shigella flexneri/isolation & purification*
Differential expression of cytokine genes in THP-1-derived macrophages infected with mild and virulence strains of Shigella flexneri 2a

Shabani NRM, Mokhtar M, Leow CH, Lean QY, Chuah C, Singh KKB, et al.

Infect Genet Evol, 2020 11;85:104532.
PMID: 32911076 DOI: 10.1016/j.meegid.2020.104532

Shigella is an intracellular bacterial pathogen that causes bacterial dysentery called shigellosis. The assessment of pro- and anti-inflammatory mediators produced by immune cells against this bacteria are vital in identifying the effectiveness of the immune reaction in protecting the host. In Malaysia, Shigella is ranked as the third most common bacteria causing diarrheal disease among children below 5 years old. In the present study, we aim to examine the differential cytokine gene expressions of macrophages in response to two types of clinical strains of Shigella flexneri 2a (S. flexneri 2a) isolated from patients admitted in Hospital Universiti Sains Malaysia, Kelantan, Malaysia. THP-1-derived macrophages, as the model of human macrophages, were infected separately with S. flexneri 2a mild (SH062) and virulence (SH057) strains for 6, 12, and 24 h, respectively. The gene expression level of inflammatory mediators was identified using real-time quantitative polymerase chain reaction (RT-qPCR). The production of nitric oxide (NO) by the macrophages was measured by using a commercialized NO assay kit. The ability of macrophages to kill the intracellular bacteria was assessed by intracellular killing assay. Induction of tumor necrosis factor-alpha (TNFα), interleukin (IL)-1β, IL-6, IL-12, inducible NO synthase (iNOS), and NO, confirmed the pro-inflammatory reaction of the THP-1-derived macrophages in response to S. flexneri 2a, especially against the SH507 strain. The SH057 also induced a marked increase in the expression levels of the anti-inflammatory cytokine mRNAs at 12 h and 24 h post-infection. In the intracellular killing assay, both strains showed less viable, indicating the generation of pro-inflammatory cytokines in the presence of iNOS and NO was crucial in the stimulation of macrophages for the host defense against shigellosis. Transcription analysis of THP-1-derived macrophages in this study identifies differentially expressed cytokine genes that correlated with the virulence factor of S. flexneri 2a.

Matched MeSH terms: Shigella flexneri/genetics*; Shigella flexneri/pathogenicity
Designing and evaluation of an antibody-targeted chimeric recombinant vaccine encoding Shigella flexneri outer membrane antigens

Kazi A, Hisyam Ismail CMK, Anthony AA, Chuah C, Leow CH, Lim BH, et al.

Infect Genet Evol, 2020 06;80:104176.
PMID: 31923724 DOI: 10.1016/j.meegid.2020.104176

Shigellosis is one of the most common diseases found in the developing countries, especially those countries that are prone flood. The causative agent for this disease is the Shigella species. This organism is one of the third most common enteropathogens responsible for childhood diarrhea. Since Shigella can survive gastric acidity and is an intracellular pathogen, it becomes difficult to treat. Also, uncontrolled use of antibiotics has led to development of resistant strains which poses a threat to public health. Therefore, there is a need for long term control of Shigella infection which can be achieved by designing a proper and effective vaccine. In this study, emphasis was made on designing a candidate that could elicit both B-cell and T-cell immune response. Hence B- and T-cell epitopes of outer membrane channel protein (OM) and putative lipoprotein (PL) from S. flexneri 2a were computationally predicted using immunoinformatics approach and a chimeric construct (chimeric-OP) containing the immunogenic epitopes selected from OM and PL was designed, cloned and expressed in E. coli system. The immunogenicity of the recombinant chimeric-OP was assessed using Shigella antigen infected rabbit antibody. The result showed that the chimeric-OP was a synthetic peptide candidate suitable for the development of vaccine and immunodiagnostics against Shigella infection.

Matched MeSH terms: Shigella flexneri/genetics; Shigella flexneri/immunology*
[Incidence of diarrhea and antibiotic therapy in cynomolgus monkeys imported for medical use (author's transl)]

Takasaka M, Morota S, Kasono T, Abe M, Honjo S

Jikken Dobutsu, 1973 Jul;22(3):227-36.
PMID: 4204642
Matched MeSH terms: Shigella flexneri/isolation & purification
Fulltext Antimicrobial activity and the presence of virulence factors and bacteriocin structural genes in Enterococcus faecium CM33 isolated from ewe colostrum

Nami Y, Haghshenas B, Haghshenas M, Yari Khosroushahi A

Front Microbiol, 2015;6:782.
PMID: 26284059 DOI: 10.3389/fmicb.2015.00782

Screening of lactic acid bacteria (LAB) isolated from ewe colostrum led to the identification and isolation of Enterococcus faecium CM33 with interesting features like high survival rates under acidic or bile salts condition, high tolerance for the simulated gastrointestinal condition, and high adhesive potential to Caco-2 cells. According the inhibition of pathogen adhesion test results, this strain can reduce more than 50% adhesion capacity of Escherichia coli, Shigella flexneri, Klebsiella pneumoniae, Listeria monocytogenes, and Staphylococcus aureus to Caco-2 cells. Based on the antibiotic sensitivity test findings, E. faecium CM33 was susceptible to gentamycin, vancomycin, erythromycin, ampicillin, penicillin, tetracycline, and rifampicin, but resistant to chloramphenicol, clindamycin, and kanamycin. Upon assessment of the virulence determinants for E. faecium CM33, this strain was negative for all tested virulence genes. Furthermore, the genome of this strain was evaluated for the incidence of the known enterocin genes by specific PCR amplification and discovered the genes encoding enterocins A, 31, X, and Q. Based on this study findings, the strain E. faecium CM33 can be considered as a valuable nutraceutical and can be introduced as a new potential probiotic.

Matched MeSH terms: Shigella flexneri