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Phage display of recombinant antibodies toward Burkholderia pseudomallei exotoxin

Nathan S, Li H, Mohamed R, Embi N

J. Biochem. Mol. Biol. Biophys., 2002 Feb;6(1):45-53.
PMID: 12186782

We have used the phagemid pComb3H to construct recombinant phages displaying the single chain variable fragment (ScFv) towards exotoxin of Burkholderia pseudomallei. Variable heavy and light chain fragments were amplified from the hybridoma 6E6A8F3B line, with a wide spectrum of primers specific to mouse antibody genes. Through overlapping extension polymerase chain reaction, the heavy and light chain fragments were linked to form the ScFv which was subsequently cloned into the phage display vector and transformed into ER2537 cells to yield a complexity of 10(8) clones. The transformants were screened by four rounds of biopanning against the exotoxin and resulted in selective enrichment of exotoxin-binding antibodies by 301 fold. The phage pool from the final round of selection displayed antibodies of high-affinity to the exotoxin as demonstrated by ELISA. Several clones were selected randomly from this pool and analysed by restriction enzyme digestion, fingerprinting and sequencing. Restriction analysis confirmed that all clones carried a 700-800 bp insert whose sequences, in general, corresponded to that of mouse IgG. Fingerprinting profiles delineated the antibodies into two families with different CDR sequences.

Matched MeSH terms: Exotoxins/immunology*
Fatal necrotizing pneumonia caused by Panton-Valentine leukocidin-producing hospital-acquired Staphylococcus aureus: a case report

Al-Talib H, Hasan H, Yean CY, Al-Ashwal SM, Ravichandran M

Jpn J Infect Dis, 2011;64(1):58-60.
PMID: 21266757

Panton-Valentine leukocidin (PVL) is a cytotoxin which causes leukocyte destruction and tissue necrosis. Although it is produced by fewer than 5% of Staphylococcus aureus strains, PVL-producing S. aureus is emerging as a serious problem worldwide. There has been a marked increase in the incidence of necrotizing lung infections with a very high mortality associated with these strains. This report describes a fatal case of hospital-acquired necrotizing pneumonia caused by PVL-positive methicillin-susceptible S. aureus in a patient with a brain tumor.

Matched MeSH terms: Exotoxins/biosynthesis*
Distribution of virulence genes and the molecular epidemiology of Streptococcus pyogenes clinical isolates by emm and multilocus sequence typing methods

Hamzah SNA, Mohd Desa MN, Jasni AS, Mohd Taib N, Masri SN, Hamat RA

Med J Malaysia, 2021 03;76(2):164-170.
PMID: 33742623

BACKGROUND: Streptococcus pyogenes has a variety of virulence factors and the predominant invasive strains differ according to specific emm types and geographical orientation. Although emm typing is commonly used as the gold standard method for the molecular characterisation, multilocus sequence typing (MLST) has become an important tool for comparing the genetic profiles globally. This study aimed to screen selected virulence genes from invasive and non-invasive clinical samples and to characterise the molecular epidemiology by emm typing and MLST methods.
MATERIALS AND METHODS: A total of 42 S. pyogenes isolates from invasive and non-invasive samples collected from two different tertiary hospitals were investigated for the distribution of virulence factors and their molecular epidemiology by emm and multilocus sequence typing methods. Detection of five virulence genes (speA, speB, speJ, ssa and sdaB) was performed using multiplex polymerase chain reaction (PCR) using the standard primers and established protocol. Phylogenetic tree branches were constructed from sequence analysis utilised by neighbour joining method generated from seven housekeeping genes using MEGA X software.
RESULTS: Multiplex PCR analysis revealed that sdaB/speF (78.6%) and speB (61.9%) were the predominant virulence genes. Regardless of the type of invasiveness, diverse distribution of emm types/subtypes was noted which comprised of 27 different emm types/subtypes. The predominant emm types/subtypes were emm63 and emm18 with each gene accounted for 11.8% whereas 12% for each gene was noted for emm28, emm97.4 and emm91. The MLST revealed that the main sequence type (ST) in invasive samples was ST402 (17.7%) while ST473 and ST318 (12% for each ST) were the major types in non-invasive samples. Out of 18 virulotypes, Virulotype A (five genes, 55.6%) and Virulotype B (two genes, 27.8%) were the major virulotypes found in this study. Phylogenetic analysis indicated the presence of seven different clusters of S. pyogenes. Interestingly, Cluster VI showed that selected emm/ST types such as emm71/ST318 (n=2), emm70.1/ST318 (n=1), emm44/ST31 (n=1) and emm18/ST442 (n=1) have clustered within a common group (Virulotype A) for both hospitals studied.
CONCLUSION: The present study showed that group A streptococcci (GAS) are genetically diverse and possess virulence genes regardless of their invasiveness. Majority of the GAS exhibited no restricted pattern of virulotypes except for a few distinct clusters. Therefore, it can be concluded that virulotyping is partially useful for characterising a heterogeneous population of GAS in hospitals.

Matched MeSH terms: Exotoxins
Staphylococcal infections and infertility: mechanisms and management

Dutta S, Sengupta P, Izuka E, Menuba I, Jegasothy R, Nwagha U

Mol Cell Biochem, 2020 Nov;474(1-2):57-72.
PMID: 32691256 DOI: 10.1007/s11010-020-03833-4

Infertility is a subject of worldwide concern as it affects approximately 15% of couples. Among the prime contributors of infertility, urogenital bacterial infections have lately gained much clinical importance. Staphylococcal species are commensal bacteria and major human pathogens mediating an array of reproductive tract infections. Emerging evidences are 'bit by bit' revealing the mechanisms by which Staphylococci strategically disrupt normal reproductive functions. Staphylococcal species can directly or through hematogenous routes can invade the reproductive tissues. In the testicular cells, epididymis as well as in various compartments of female reproductive tracts, the pathogen recognition receptors, toll-like receptors (TLRs), can recognize the pathogen-associated molecular patterns on the Staphylococci and thereby activate inflammatory signalling pathways. These elicit pro-inflammatory mediators trigger other immune cells to infiltrate and release further inflammatory agents and reactive oxygen species (ROS). Adaptive immune responses may intensify the inflammation-induced reproductive tissue damage, particularly via activation of T-helper (Th) cells, Th1 and Th17 by the innate components or by staphylococcal exotoxins. Staphylococcal surface factors binding with sperm membrane proteins can directly impair sperm functions. Although Staphylococci, being one of the most virulent bacterial species, are major contributors in infection-induced infertility in both males and females, the mechanisms of their operations remain under-discussed. The present review aims to provide a comprehensive perception of the possible mechanisms of staphylococcal infection-induced male and female infertility and aid potential interventions to address the lack of competent therapeutic measures for staphylococcal infection-induced infertility.

Matched MeSH terms: Exotoxins
Fulltext Frequency and molecular epidemiology of Panton-Valentine leukocidin gene in Staphylococcus aureus colonising HIV-infected patients

Mohd-Zain Z, Mohd-Nawi SFA, Adnan A, Kumar S

Malays J Pathol, 2017 Aug;39(2):115-122.
PMID: 28866692 MyJurnal

BACKGROUND: HIV-infected patients pose a high risk of contracting skin and soft tissue infections caused by Staphylococcus aureus. Those who are colonized with methicillin-resistant S. aureus (MRSA) that carry Panton-Valentine leukocidin (PVL) are predisposed to severe infections that could lead to necrotic skin infections. However the association of S. aureus specifically methicillin sensitive S. aureus carrying PVL gene in HIV patients has not been widely reported. Here, we study the prevalence and the molecular epidemiology of PVL-producing S. aureus in HIV-infected patients.
METHODS: Swabs from four body sites of 129 HIV-infected patients were cultured for S. aureus and identified by standard microbiological procedures. The isolates were subjected to antimicrobial susceptibility testing by disk diffusion against penicillin, erythromycin, clindamycin, and cotrimoxazole. PCR was used to detect the PVL gene and genetic relationship between the isolates was determined by using pulse field gel electrophoresis.
RESULTS: A total of 51 isolates of S. aureus were obtained from 40 (31%) of the patients. The majority (43.1%) of the isolates were obtained from the anterior nares. Thirteen (25.5%) of all the isolates were resistant to more than one category of antibiotics, with one isolate identified as MRSA. Thirty-eight (74.5%) isolates (including the MRSA isolate) carried PVL gene where the majority (44.7%) of these isolates were from the anterior nares. A dendogram revealed that the isolates were genetically diverse with 37 distinct pulsotypes clustered in 11 groups.
CONCLUSION: S. aureus obtained from multiple sites of the HIV patients were genetically diverse without any clonality observed.

Matched MeSH terms: Exotoxins
Fulltext Origin and evolution of European community-acquired methicillin-resistant Staphylococcus aureus

Stegger M, Wirth T, Andersen PS, Skov RL, De Grassi A, Simões PM, et al.

mBio, 2014 Aug 26;5(5):e01044-14.
PMID: 25161186 DOI: 10.1128/mBio.01044-14

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) was recognized in Europe and worldwide in the late 1990s. Within a decade, several genetically and geographically distinct CA-MRSA lineages carrying the small SCCmec type IV and V genetic elements and the Panton-Valentine leukocidin (PVL) emerged around the world. In Europe, the predominant CA-MRSA strain belongs to clonal complex 80 (CC80) and is resistant to kanamycin/amikacin and fusidic acid. CC80 was first reported in 1993 but was relatively rare until the late 1990s. It has since been identified throughout North Africa, the Middle East, and Europe, with recent sporadic reports in sub-Saharan Africa. While strongly associated with skin and soft tissue infections, it is rarely found among asymptomatic carriers. Methicillin-sensitive S. aureus (MSSA) CC80 strains are extremely rare except in sub-Saharan Africa. In the current study, we applied whole-genome sequencing to a global collection of both MSSA and MRSA CC80 isolates. Phylogenetic analyses strongly suggest that the European epidemic CA-MRSA lineage is derived from a PVL-positive MSSA ancestor from sub-Saharan Africa. Moreover, the tree topology suggests a single acquisition of both the SCCmec element and a plasmid encoding the fusidic acid resistance determinant. Four canonical SNPs distinguish the derived CA-MRSA lineage and include a nonsynonymous mutation in accessory gene regulator C (agrC). These changes were associated with a star-like expansion into Europe, the Middle East, and North Africa in the early 1990s, including multiple cases of cross-continent imports likely driven by human migrations.
IMPORTANCE: With increasing levels of CA-MRSA reported from most parts of the Western world, there is a great interest in understanding the origin and factors associated with the emergence of these epidemic lineages. To trace the origin, evolution, and dissemination pattern of the European CA-MRSA clone (CC80), we sequenced a global collection of strains of the S. aureus CC80 lineage. Our study determined that a single descendant of a PVL-positive methicillin-sensitive ancestor circulating in sub-Saharan Africa rose to become the dominant CA-MRSA clone in Europe, the Middle East, and North Africa. In the transition from a methicillin-susceptible lineage to a successful CA-MRSA clone, it simultaneously became resistant to fusidic acid, a widely used antibiotic for skin and soft tissue infections, thus demonstrating the importance of antibiotic selection in the success of this clone. This finding furthermore highlights the significance of horizontal gene acquisitions and underscores the combined importance of these factors for the success of CA-MRSA.

Matched MeSH terms: Exotoxins/genetics
Expression and purification of a recombinant scFv towards the exotoxin of the pathogen, Burkholderia pseudomallei

Lim KP, Li H, Nathan S

J Microbiol, 2004 Jun;42(2):126-32.
PMID: 15357306

A single chain variable fragment (scFv) specific towards B. pseudomallei exotoxin had previously been generated from an existing hybridoma cell line (6E6AF83B) and cloned into the phage display vector pComb3H. In this study, the scFv was subcloned into the pComb3X vector to facilitate the detection and purification of expressed antibodies. Detection was facilitated by the presence of a hemagglutinin (HA) tag, and purification was facilitated by the presence of a histidine tag. The culture was grown at 30 degrees C until log phase was achieved and then induced with 1 mM IPTG in the absence of any additional carbon source. Induction was continued at 30 degrees C for five h. The scFv was discerned by dual processes-direct enzyme-linked immunosorbent assays (ELISA), and Western blotting. When compared to E. coli strains ER2537 and HB2151, scFv expression was observed to be highest in the E. coli strain Top10F'. The expressed scFv protein was purified via nickel-mediated affinity chromatography and results indicated that two proteins a 52 kDa protein, and a 30 kDa protein were co-purified. These antibodies, when blotted against immobilized exotoxin, exhibited significant specificity towards the exotoxin, compared to other B. pseudomallei antigens. Thus, these antibodies should serve as suitable reagents for future affinity purification of the exotoxin.

Matched MeSH terms: Exotoxins/immunology*
Fulltext Characteristics, detection and typing methods of community acquired methicillin resistant staphylococcus aureus ((CA-MRSA) – a review

Lia Natasha Amit, John DV, Fong SM

Malaysian Journal of Public Health Medicine, 2017;17(3):27-37.

Staphylococcus aureus aregram positive cocci which colonizethe skin and mucous membranes particularly the anterior nares. Prevalence of nosocomial infections associated with methicillin resistant S. aureus have been reported in hospitals (HA-MRSA) for over five decades. Recently,community-acquired MRSA (CA-MRSA) has emerged as a cause of skin and soft tissue infections in healthy individuals. These strains are sensitive to antimicrobials, carry genes for Panton-Valentine leukocidin (PVL) toxin and belong to the staphylococcal cassette chromosome (SCC) mec type IV or V. The suspected mode of transmission involves close contact with carriers leading to skin or nasal colonization that resultin subsequent active infection. Molecular typing is used to determine the mode of transmission of CA-MRSA in the community.General typing methods such as pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) and specific methods for Staphylococci such as SCCmec typing and spa typing have the capability to characterize bacterial chromosomes and mobile genetic elements. Combination of these molecular typing methods is necessary as each method has its own advantages with respect to discriminatory power, rapidity, cost effectiveness, reproducibility, and ease of performance.

Matched MeSH terms: Exotoxins