The effect of human amniotic epithelial cells on urethral stricture fibroblasts.

BACKGROUND: Urethral stricture disease (USD) is effectively managed by buccal mucosa (BM) urethroplasty. Lack of adequate healthy BM has led to the use of autologous tissue-engineered BM grafts. Such grafts are costly, not easily scalable and recurrence of the stricture is still a problem. Hence, there is a requirement for cost-effective, scalable cells with innate antifibrotic properties which seem to be fulfilled by human amniotic epithelial cells (HAMECs). The effect of HAMECs on USD is unknown. AIM: To study the effect of HAMECs-CM on human urethral stricture fibroblast (USF) cells by using in-vitro migration assay and molecular techniques. MATERIALS AND METHODS: USF cells were derived from six patients undergoing urethroplasty. HAMECs were derived from one placenta after delivery. The effect of HAMECs-CM on USF cell migration was observed using a standard in vitro scratch assay over a period of 3 days. The effect of HAMECs-CM on the expression levels of markers alpha-smooth muscle actin (α-SMA) and tissue inhibitor of metalloproteinases (TIMP-1) in USF cells was also examined. RESULTS: The HAMECs-CM suppressed the migration of USF cells in in vitro scratch assay. The HAMECs-CM consistently downregulated α-SMA, but not TIMP-1. CONCLUSIONS: HAMECs have shown antifibrotic activity on USF cells in this in vitro study. RELEVANCE FOR PATIENTS: HAMECs could serve as an alternative cell source for tissue-engineered urethroplasty.

Cloning and high-level expression of Thermus thermophilus RecA in E. coli: purification and novel use in HBV diagnostics

ABSTRACT We studied the role of Thermus thermophilus Recombinase A (RecA) in enhancing the PCR signals of DNA viruses such as Hepatitis B virus (HBV). The RecA gene of a thermophilic eubacterial strain, T. thermophilus, was cloned and hyperexpressed in Escherichia coli. The recombinant RecA protein was purified using a single heat treatment step without the use of any chromatography steps, and the purified protein (>95%) was found to be active. The purified RecA could enhance the polymerase chain reaction (PCR) signals of HBV and improve the detection limit of the HBV diagnosis by real time PCR. The yield of recombinant RecA was ∼35 mg/L, the highest yield reported for a recombinant RecA to date. RecA can be successfully employed to enhance detection sensitivity for the diagnosis of DNA viruses such as HBV, and this methodology could be particularly useful for clinical samples with HBV viral loads of less than 10 IU/mL, which is interesting and novel.

Novel properties of recombinant Sso7d-Taq DNA polymerase purified using aqueous two-phase extraction: Utilities of the enzyme in viral diagnosis.

Using Sso7d from Sulfolobus solfataricus as the DNA binding protein fused to Taq DNA polymerase at its amino terminus, we report the hyper-expression and a novel purification methodology of Sso7d-Taq polymerase (S-Taq) using aqueous two-phase extraction system followed by Ni-affinity chromatography. The utility of such a fusion enzyme in carrying out PCR of human genes from whole blood directly and in detecting hepatitis B virus from clinical samples is demonstrated in this article. We present data on the enhanced thermo-stability of S-Taq DNA polymerase over Taq DNA polymerase and also provide evidence of its higher stability with detergents in comparison to Taq polymerase. The purified S-Taq protein showed acceptable limits of host genomic DNA levels without the use of DNases and other DNA precipitating agents and shows promising potential for use in PCR based diagnostics, in-situ PCR's and forensic science.

Use of coagulation factor XIII (F13) gene as an internal control for normalization of genomic DNA's for HLA typing.

Genomic DNA (gDNA) obtained from whole blood samples is a critical element for genomic research and clinical diagnosis. PCR efficiencies of the targeted genes like HLA-A, -B, -C, DPB1 and DRB1 using such isolated gDNAs were variable in spite of having similar amounts of gDNA taken for PCR. We addressed such PCR variabilities by normalizing the gDNA's using an internal control of human coagulation factor XIII that was found to be variable with all samples and did not correlate with the observed A260 nm readings. The PCR and Q-PCR methodologies for the human coagulation factor XIII have been optimized, and the advantages of normalizing gDNA preparations based on F13 copy numbers have been discussed. This method will serve as a suitable choice to be used in laboratories and research centres, particularly when dealing with a large number of samples for the next-generation sequencing purposes, and in forensic labs with limited sample availability.

Buccal Mucosal Epithelial Cells Downregulate CTGF Expression in Buccal Submucosal Fibrosis Fibroblasts.

INTRODUCTION: Oral submucosal fibrosis (OSMF) is a chronic debilitating fibrotic disease of the oral cavity and is a serious health hazard in south Asia and, increasingly, the rest of the world. The molecular basis behind various treatment modalities to treat OSMF still remains unclear. In this study, we have investigated the in vitro ability of the buccal mucosal cells to reduce the proliferation of the fibroblasts of the fibrotic area in co-culture of cells and also at the molecular levels to reduce the level of connective tissue growth factor (CTGF) in the OSMF fibroblasts (SMF-F). MATERIALS AND METHODS: The study compares isolation, morphological and proliferation kinetics of SMF-F and BMF cells with and without co-culturing with BMEs. In addition, we have compared the mRNA expression levels of CTGF in SMF-F co-cultured BME and non-co-cultured SMF-F cells using validated real-time quantitative PCR (RT-qPCR) method. RESULTS: The basic morphological characteristics of SMF-F were similar to BMF, but the former cells had higher proliferation rate in early passages compared to late passage state. We also observed that the CTGF expression levels in SMF-F under co-culture conditions of BME were consistently and significantly downregulated in all four different SMF-F-derived cells from four different patients. CONCLUSION: Rapid proliferation and collagen synthesis in SMF-F as against BMF cells are the factors that confirm the innate nature of fibrosis fibroblasts (SMF-F). Further, the CTGF expression level in SMF-F was significantly suppressed by BME in co-culture conditions against controls (BMF). Considered together, this suggests that the cell therapeutic candidate of BME could be used in treating OSMF.

Cloning and high-level expression of Thermus thermophilus RecA in E. coli: purification and novel use in HBV diagnostics.

We studied the role of Thermus thermophilus Recombinase A (RecA) in enhancing the PCR signals of DNA viruses such as Hepatitis B virus (HBV). The RecA gene of a thermophilic eubacterial strain, T. thermophilus, was cloned and hyperexpressed in Escherichia coli. The recombinant RecA protein was purified using a single heat treatment step without the use of any chromatography steps, and the purified protein (>95%) was found to be active. The purified RecA could enhance the polymerase chain reaction (PCR) signals of HBV and improve the detection limit of the HBV diagnosis by real time PCR. The yield of recombinant RecA was ∼35mg/L, the highest yield reported for a recombinant RecA to date. RecA can be successfully employed to enhance detection sensitivity for the diagnosis of DNA viruses such as HBV, and this methodology could be particularly useful for clinical samples with HBV viral loads of less than 10IU/mL, which is interesting and novel.

Bacteriophage-derived CHAP domain protein, P128, kills Staphylococcus cells by cleaving interpeptide cross-bridge of peptidoglycan.

P128 is an anti-staphylococcal protein consisting of the Staphylococcus aureus phage-K-derived tail-associated muralytic enzyme (TAME) catalytic domain (Lys16) fused with the cell-wall-binding SH3b domain of lysostaphin. In order to understand the mechanism of action and emergence of resistance to P128, we isolated mutants of Staphylococcus spp., including meticillin-resistant Staphylococcus aureus (MRSA), resistant to P128. In addition to P128, the mutants also showed resistance to Lys16, the catalytic domain of P128. The mutants showed loss of fitness as shown by reduced rate of growth in vitro. One of the mutants tested was found to show reduced virulence in animal models of S. aureus septicaemia suggesting loss of fitness in vivo as well. Analysis of the antibiotic sensitivity pattern showed that the mutants derived from MRSA strains had become sensitive to meticillin and other ß-lactams. Interestingly, the mutant cells were resistant to the lytic action of phage K, although the phage was able to adsorb to these cells. Sequencing of the femA gene of three P128-resistant mutants showed either a truncation or deletion in femA, suggesting that improper cross-bridge formation in S. aureus could be causing resistance to P128. Using glutathione S-transferase (GST) fusion peptides as substrates it was found that both P128 and Lys16 were capable of cleaving a pentaglycine sequence, suggesting that P128 might be killing S. aureus by cleaving the pentaglycine cross-bridge of peptidoglycan. Moreover, peptides corresponding to the reported cross-bridge of Staphylococcus haemolyticus (GGSGG, AGSGG), which were not cleaved by lysostaphin, were cleaved efficiently by P128. This was also reflected in high sensitivity of S. haemolyticus to P128. This showed that in spite of sharing a common mechanism of action with lysostaphin, P128 has unique properties, which allow it to act on certain lysostaphin-resistant Staphylococcus strains.

Properties and mutation studies of a bacteriophage-derived chimeric recombinant staphylolytic protein P128: Comparison to recombinant lysostaphin.

P128 is a chimeric anti-staphylococcal protein having a catalytic domain from a Staphylococcus bacteriophage K tail associated structural protein and a cell wall targeting domain from the Staphylococcus bacteriocin-lysostaphin. In this study, we disclose additional properties of P128 and compared the same with lysostaphin. While lysostaphin was found to get inactivated by heat and was inactive on its parent strain S. simulans biovar staphylolyticus, P128 was thermostable and was lytic towards S. simulans biovar staphylolyticus demonstrating a difference in their mechanism of action. Selected mutation studies of the catalytic domain of P128 showed that arginine and cysteine, at 40th and 76th positions respectively, are critical for the staphylolytic activity of P128, although these amino acids are not conserved residues. In comparison to native P128, only the R40S mutant (P301) was catalytically active on zymogram gel and had a similar secondary structure, as assessed by circular dichroism analysis and in silico modeling with similar cell binding properties. Mutation of the arginine residue at 40th position of the P128 molecule caused dramatic reduction in the Vmax (∆OD600 [mg/min]) value (nearly 270 fold) and the recombinant lysostaphin also showed lesser Vmax value (nearly 1.5 fold) in comparison to the unmodified P128 protein. The kinetic parameters such as apparent Km (KmAPP) and apparent Kcat (KcatAPP) of the native P128 protein also showed significant differences in comparison to the values observed for P301 and lysostaphin.

Use of prophage free host for achieving homogenous population of bacteriophages: new findings.

We demonstrate that the prophage status of bacteria plays a critical role in achieving homogenous population of a phage preparation. When a lytic Staphylococcus bacteriophage 44AHJD was propagated in a Staphylococcus clinical isolate, the enriched phage showed 44AHJD phage virions along with the released prophages from the baiting host. The released prophage was identified as a siphophage by transmission electron microscopy. To obtain a phage preparation free of prophages, when we carried out multiplication of the 44AHJD phage in a prophage free Staphyloccoccus aureus host namely RN4220, we were surprised not to see any phage plaques in spite of the phage exhibiting >99.9% adsorption to such cells. Since RN4220 host is devoid of restriction modification system and prophages, we hypothesized that in spite of successful infection and multiplication, the phage virions might have failed to show plaques due to its insignificant release from the cell possibly due to insufficient endolysin expressed from phage virions during phage development and assembly. Our hypothesis was confirmed when we observed plaques of 44AHJD phage in RN4220 cells where additional phage endolysin protein was supplemented via a plasmid. Endolysin protein from various types of Staphylococcus phages showed plaques of 44AHJD in RN4220 cells confirming our hypothesis. Also, we demonstrate for the first time that propagation of 44AHJD phage with endolysin supplementation in prophage free RN4220 host yields pure phage preparation.

Staphylococcus bacteriophage tails with bactericidal properties: new findings.

The development of lytic bacteriophages as therapeutic products is an attractive alternative to antibiotics. In this study, we evaluated the potential of phage tails for lysing Gram-positive bacteria. Phage P954, a well-characterized temperate staphylococcal phage, was found to adsorb to a large number of Staphylococcus aureus clinical isolates, although it lyses only 24% of the tested isolates. However, P954 phage tails generated by interruption of phage assembly were bactericidal against all the phage-resistant isolates. Phage tail preparations were trypsin sensitive with an apparent molecular weight of over 300 kDa. PCR analysis of the P954 phage-resistant isolates indicated the integration of P954-like prophages into the host genomes. Our study demonstrates for the first time that P954 bacteriophage tails have a much broader host range than the intact phage because phage tails are not affected by superinfection immunity or vulnerable to host restriction endonucleases.

A novel bacteriophage Tail-Associated Muralytic Enzyme (TAME) from Phage K and its development into a potent antistaphylococcal protein.

BACKGROUND: Staphylococcus aureus is a major cause of nosocomial and community-acquired infections. However, the rapid emergence of antibiotic resistance limits the choice of therapeutic options for treating infections caused by this organism. Muralytic enzymes from bacteriophages have recently gained attention for their potential as antibacterial agents against antibiotic-resistant gram-positive organisms. Phage K is a polyvalent virulent phage of the Myoviridae family that is active against many Staphylococcus species. RESULTS: We identified a phage K gene, designated orf56, as encoding the phage tail-associated muralytic enzyme (TAME). The gene product (ORF56) contains a C-terminal domain corresponding to cysteine, histidine-dependent amidohydrolase/peptidase (CHAP), which demonstrated muralytic activity on a staphylococcal cell wall substrate and was lethal to S. aureus cells. We constructed N-terminal truncated forms of ORF56 and arrived at a 16-kDa protein (Lys16) that retained antistaphylococcal activity. We then generated a chimeric gene construct encoding Lys16 and a staphylococcal cell wall-binding SH3b domain. This chimeric protein (P128) showed potent antistaphylococcal activity on global clinical isolates of S. aureus including methicillin-resistant strains. In addition, P128 was effective in decolonizing rat nares of S. aureus USA300 in an experimental model. CONCLUSIONS: We identified a phage K gene that encodes a protein associated with the phage tail structure. The muralytic activity of the phage K TAME was localized to the C-terminal CHAP domain. This potent antistaphylococcal TAME was combined with an efficient Staphylococcus-specific cell-wall targeting domain SH3b, resulting in the chimeric protein P128. This protein shows bactericidal activity against globally prevalent antibiotic resistant clinical isolates of S. aureus and against the genus Staphylococcus in general. In vivo, P128 was efficacious against methicillin-resistant S. aureus in a rat nasal colonization model.

Lysis-deficient phages as novel therapeutic agents for controlling bacterial infection.

BACKGROUND: Interest in phage therapy has grown over the past decade due to the rapid emergence of antibiotic resistance in bacterial pathogens. However, the use of bacteriophages for therapeutic purposes has raised concerns over the potential for immune response, rapid toxin release by the lytic action of phages, and difficulty in dose determination in clinical situations. A phage that kills the target cell but is incapable of host cell lysis would alleviate these concerns without compromising efficacy. RESULTS: We developed a recombinant lysis-deficient Staphylococcus aureus phage P954, in which the endolysin gene was rendered nonfunctional by insertional inactivation. P954, a temperate phage, was lysogenized in S. aureus strain RN4220. The native endolysin gene on the prophage was replaced with an endolysin gene disrupted by the chloramphenicol acetyl transferase (cat) gene through homologous recombination using a plasmid construct. Lysogens carrying the recombinant phage were detected by growth in presence of chloramphenicol. Induction of the recombinant prophage did not result in host cell lysis, and the phage progeny were released by cell lysis with glass beads. The recombinant phage retained the endolysin-deficient genotype and formed plaques only when endolysin was supplemented. The host range of the recombinant phage was the same as that of the parent phage. To test the in vivo efficacy of the recombinant endolysin-deficient phage, immunocompromised mice were challenged with pathogenic S. aureus at a dose that results in 80% mortality (LD80). Treatment with the endolysin-deficient phage rescued mice from the fatal S. aureus infection. CONCLUSIONS: A recombinant endolysin-deficient staphylococcal phage has been developed that is lethal to methicillin-resistant S. aureus without causing bacterial cell lysis. The phage was able to multiply in lytic mode utilizing a heterologous endolysin expressed from a plasmid in the propagation host. The recombinant phage effectively rescued mice from fatal S. aureus infection. To our knowledge this is the first report of a lysis-deficient staphylococcal phage.