Genomic characterization and virulence of Streptococcus suis serotype 4 clonal complex 94 recovered from human and swine samples.

Streptococcus suis is a zoonotic pathogen that causes invasive infections in humans and pigs. Herein, we performed genomic analysis of seven S. suis serotype 4 strains belonging to clonal complex (CC) 94 that were recovered from a human patient or from diseased and clinically healthy pigs. Genomic exploration and comparisons, as well as in vitro cytotoxicity tests, indicated that S. suis CC94 serotype 4 strains are potentially virulent. Genomic analysis revealed that all seven strains clustered within minimum core genome group 3 (MCG-3) and had a high number of virulence-associated genes similar to those of virulent serotype 2 strains. Cytotoxicity assays showed that both the human lung adenocarcinoma cell line and HeLa cells rapidly lost viability following incubation for 4 h with the strains at a concentration of 106 bacterial cells. The human serotype 4 strain (ID36054) decreased cell viability profoundly and similarly to the control serotype 2 strain P1/7. In addition, strain ST1689 (ID34572), isolated from a clinically healthy pig, presented similar behaviour in an adenocarcinoma cell line and HeLa cells. The antimicrobial resistance genes tet(O) and ermB that confer resistance to tetracyclines, macrolides, and lincosamides were commonly found in the strains. However, aminoglycoside and streptothricin resistance genes were found only in certain strains in this study. Our results indicate that S. suis CC94 serotype 4 strains are potentially pathogenic and virulent and should be monitored.

Vitamin D (1α,25(OH)2D3) supplementation minimized multinucleated giant cells formation and inflammatory response during Burkholderia pseudomallei infection in human lung epithelial cells.

Melioidosis is an infectious disease with high mortality rates in human, caused by the bacterium Burkholderia pseudomallei. As an intracellular pathogen, B. pseudomallei can escape from the phagosome and induce multinucleated giant cells (MNGCs) formation resulting in antibiotic resistance and immune evasion. A novel strategy to modulate host response against B. pseudomallei pathogenesis is required. In this study, an active metabolite of vitamin D3 (1α,25-dihydroxyvitamin D3 or 1α,25(OH)2D3) was selected to interrupt pathogenesis of B. pseudomallei in a human lung epithelium cell line, A549. The results demonstrated that pretreatment with 10-6 M 1α,25(OH)2D3 could reduce B. pseudomallei internalization to A549 cells at 4 h post infection (P < 0.05). Interestingly, the presence of 1α,25(OH)2D3 gradually reduced MNGC formation at 8, 10 and 12 h compared to that of the untreated cells (P < 0.05). Furthermore, pretreatment with 10-6 M 1α,25(OH)2D3 considerably increased hCAP-18/LL-37 mRNA expression (P < 0.001). Additionally, pro-inflammatory cytokines, including MIF, PAI-1, IL-18, CXCL1, CXCL12 and IL-8, were statistically decreased (P < 0.05) in 10-6 M 1α,25(OH)2D3-pretreated A549 cells by 12 h post-infection. Taken together, this study indicates that pretreatment with 10-6 M 1α,25(OH)2D3 has the potential to reduce the internalization of B. pseudomallei into host cells, decrease MNGC formation and modulate host response during B. pseudomallei infection by minimizing the excessive inflammatory response. Therefore, 1α,25(OH)2D3 supplement may provide an effective supportive treatment for melioidosis patients to combat B. pseudomallei infection and reduce inflammation in these patients.

D-LL-31 enhances biofilm-eradicating effect of currently used antibiotics for chronic rhinosinusitis and its immunomodulatory activity on human lung epithelial cells.

Chronic rhinosinusitis (CRS) is a chronic disease that involves long-term inflammation of the nasal cavity and paranasal sinuses. Bacterial biofilms present on the sinus mucosa of certain patients reportedly exhibit resistance against traditional antibiotics, as evidenced by relapse, resulting in severe disease. The aim of this study was to determine the killing activity of human cathelicidin antimicrobial peptides (LL-37, LL-31) and their D-enantiomers (D-LL-37, D-LL-31), alone and in combination with conventional antibiotics (amoxicillin; AMX and tobramycin; TOB), against bacteria grown as biofilm, and to investigate the biological activities of the peptides on human lung epithelial cells. D-LL-31 was the most effective peptide against bacteria under biofilm-stimulating conditions based on IC50 values. The synergistic effect of D-LL-31 with AMX and TOB decreased the IC50 values of antibiotics by 16-fold and could eliminate the biofilm matrix in all tested bacterial strains. D-LL-31 did not cause cytotoxic effects in A549 cells at 25 µM after 24 h of incubation. Moreover, a cytokine array indicated that there was no significant induction of the cytokines involving in immunopathogenesis of CRS in the presence of D-LL-31. However, a tissue-remodeling-associated protein was observed that may prevent the progression of nasal polyposis in CRS patients. Therefore, a combination of D-LL-31 with AMX or TOB may improve the efficacy of currently used antibiotics to kill biofilm-embedded bacteria and eliminate the biofilm matrix. This combination might be clinically applicable for treatment of patients with biofilm-associated CRS.

Promising Anticancer Effect of Aurisin A Against the Human Lung Cancer A549 Cell Line.

OBJECTIVE: To investigate the anticancer effect of aurisin A and the underlying mechanisms of its action on the human lung cancer A549 cell line. METHODS: Cell viability was determined by sulforhodamine B (SRB) assay, while cell cycle distribution and apoptosis were measured by flow cytometry. The molecular underlying mechanisms of anti-cancer properties of aurisin A was determined by western blot analysis. RESULTS: Aurisin A exerts its anticancer effects by inhibiting cell growth (p<0.001), increasing the proportion of cells at the G0/G1 phase (p<0.001), and decreasing the expression of cyclin D (p<0.05) and cyclin-dependent kinase 4 (Cdk-4) (p<0.001). Nuclear morphological changes were observed in aurisin A-treated cells, demonstrated by a dose-dependent increase in the number of apoptosis cells (p<0.001). After aurisin A treatment, B-cell lymphoma 2 (Bcl-2) was down-regulated (p<0.05), cleaved caspase-3 was up-regulated (p<0.05). In addition, aurisin A inhibits migration of cancer cells in a dose-dependent manner (p<0.001) and decreases the expression of epidermal growth factor receptor (EGFR) (p<0.05) and phosphorylated p38 (pp38) (p<0.05). CONCLUSION: These results indicated that in-vitro treatment of aurisin A against this human lung cancer cell line inhibits cell proliferation and migration, and induces apoptosis and cell-cycle arrest.  Aurisin A is a promising anticancer agent for use against human lung cancer.
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A predicted cation transporter protein, BPSS1228, is involved in intracellular behaviour of Burkholderia pseudomallei in a human lung epithelial cell line (A549).

Burkholderia pseudomallei causes melioidosis, a potentially fatal infectious disease in tropical and subtropical countries worldwide. The intracellular behaviour of this pathogen in host cells has been reported to impact the severity of melioidosis, including the development of septicaemia, a consequence of pneumonia melioidosis. We previously identified a predicted cation transporter protein, BPSS1228, that participates in the transitional stage of this intracellular pathogen. For further analysis, in this study B. pseudomallei bpss1228 mutant and complemented strains were constructed and bacterial infectivity on human lung epithelial cells, A549, investigated in vitro Burkholderia pseudomallei bpss1228 mutant showed impaired bacterial adhesion and invasion into A549 cells compared with wild-type strain, while the deficient phenotypes were restored to wild-type levels by the complemented strain. Additionally, the inactivation of bpss1228 in the mutant strain affected flagella-based swimming on a semi-solid surface and resistance to acid stresses simulating intracellular environments. These observations of BPSS1228 relating to B. pseudomallei infection strategies shed a new light on its association with intracellular B. pseudomallei during the interaction with host cells.

Transcription level analysis of intracellular Burkholderia pseudomallei illustrates the role of BPSL1502 during bacterial interaction with human lung epithelial cells.

Melioidosis caused by Burkholderia pseudomallei is a globally important disease of increasing concern according to high case-fatality rate and epidemic spreading. The ability of B. pseudomallei to attach and invade host cells and subsequently survive intracellularly has stimulated many questions concerning the comprehension of bacterial pathogenesis progression. Transcription levels of intracellular B. pseudomallei genes in human lung epithelial cells were therefore analyzed using bioinformatic tools, RT-PCR and real time RT-PCR. Here, it is reported that the identification of bpsl1502, encoding B. pseudomallei SurE (stationary phase survival protein E) located in a global transcriptional regulation operon was accomplished. The up-regulation of B. pseudomallei SurE was demonstrated during intracellular survival of A549 cells at 12, 18, and 24 h post-infection. To investigate the role of this protein, a B. pseudomallei SurE defective mutant was constructed. The invasion and initial survival of the SurE mutants within the A549 cells were impaired. There was no difference, however, between the growth of B. pseudomallei SurE mutant as compared to the wild type in Luria-Bertani culture. These data suggest that SurE may assist B. pseudomallei host cells invade and facilitate early intracellular infection but is not crucial during the stationary growth phase. The identification of B. pseudomallei SurE provides more information of bacterial strategy during an early step of the pathogenesis process of melioidosis.

Role of Burkholderia pseudomallei in the invasion, replication and induction of apoptosis in human epithelial cell lines.

Burkholderia pseudomallei is a bacterial pathogen causing melioidosis. In the present study, we demonstrated the effects of B. pseudomallei capsular polysaccharide on the invasion, intracellular replication, induction of cytotoxicity and apoptosis of human epithelial HeLa and A549 cells. The B. pseudomallei capsule mutant had a significantly greater ability to invade both cell lines (p < 0.05). The B. pseudomallei capsule mutant had a greater ability to induce apoptosis in A549 cells than wild type B. pseudomallei. These results indicate the capsular polysaccharide of B. pseudomallei plays an important role in inhibiting invasion by the bacteria but has no effect on intracellular multiplication, induction of cytotoxicity or apoptosis in epithelial cells.