The type III secretion system is involved in Escherichia coli K1 interactions with Acanthamoeba

The type III secretion system among Gram-negative bacteria is known to deliver effectors into host cell to interfere with host cellular processes. The type III secretion system in Yersina, Pseudomonas and Enterohemorrhagic Escherichia coli have been well documented to be involved in the bacterial pathogenicity. The existence of type III secretion system has been demonstrated in neuropathogenic E. coli K1 strains. Here, it is observed that the deletion mutant of type III secretion system in E. coli strain EC10 exhibited defects in the invasion and intracellular survival in Acanthamoeba castellanii (a keratitis isolate) compared to its parent strain. Next, it was determined whether type III secretion system plays a role in E. coli K1 survival inside Acanthamoeba during the encystment process. Using encystment assays, our findings revealed that the type III secretion system-deletion mutant exhibited significantly reduced survival inside Acanthamoeba cysts compared with its parent strain, EC10 (P < 0.01). This is the first demonstration that the type III secretion system plays an important role in E. coli interactions with Acanthamoeba. A complete understanding of how amoebae harbor bacterial pathogens will help design strategies against E. coli transmission to the susceptible hosts.     

Absolute Configuration of Oplopanone Derivatives From Serphidium stenocephalum: ECD Spectra of Acyclic Ketones With Front‐Octant Contributions

The electronic circular dichroism (ECD) spectra of two sesquiterpenoids ( 1 and 2 ) related to oplopanone, obtained from a methanolic extract of the plant Serphidium stenocephalum (Artemisia stenocephala), were measured and reproduced by means of time‐dependent density functional theory (TDDFT) calculations, establishing their absolute configuration. The application of ketone octant rule for carbonyl n‐π* ECD band to compounds 1 and 2 , which include an acyclic carbonyl group, was critically assessed. The peculiar oplopanone skeleton makes a straightforward application of the octant rule impossible, because of the uncertainty related to the shape of the so‐called third nodal surface separating front and back octants. The various group contributions to the carbonyl n‐π* ECD band were estimated with TDDFT calculations on selected molecular models obtained by consecutive dissections from 1 . Chirality 26:39–43, 2013. © 2013 Wiley Periodicals, Inc.     

Infection with Host-Range Mutant Adenovirus 5 Suppresses Innate Immunity and Induces Systemic CD4+ T Cell Activation in Rhesus Macaques

Ad5 is a common cause of respiratory disease and an occasional cause of gastroenteritis and conjunctivitis, and seroconversion before adolescence is common in humans. To gain some insight into how Ad5 infection affects the immune system of rhesus macaques (RM) 18 RM were infected with a host-range mutant Ad5 (Ad5hr) by 3 mucosal inoculations. There was a delay of 2 to 6 weeks after the first inoculation before plasmacytoid dendritic cell (pDC) frequency and function increased in peripheral blood. Primary Ad5hr infection suppressed IFN-γ mRNA expression, but the second Ad5hr exposure induced a rapid increase in IFN-gamma mRNA in peripheral blood mononuclear cells (PBMC). Primary Ad5hr infection suppressed CCL20, TNF and IL-1 mRNA expression in PBMC, and subsequent virus exposures further dampened expression of these pro-inflammatory cytokines. Primary, but not secondary, Ad5hr inoculation increased the frequency of CXCR3+ CD4+ T cells in blood, while secondary, but not primary, Ad5hr infection transiently increased the frequencies of Ki67+, HLADR+ and CD95+/CCR5+ CD4+ T cells in blood. Ad5hr infection induced polyfunctional CD4 and CD8+ T cells specific for the Ad5 hexon protein in all of the animals. Thus, infection with Ad5hr induced a complex pattern of innate and adaptive immunity in RM that included transient systemic CD4+ T cell activation and suppressed innate immunity on re-exposure to the virus. The complex effects of adenovirus infection on the immune system may help to explain the unexpected results of testing Ad5 vector expressing HIV antigens in Ad5 seropositive people.     

Sentinel Hospital-Based Surveillance for Assessment of Burden of Rotavirus Gastroenteritis in Children in Pakistan

Objectives

To determine the burden and molecular epidemiology of rotavirus gastroenteritis in children hospitalized with severe acute watery diarrhea in Pakistan prior to introduction of rotavirus vaccine.

Methods

A cross-sectional study was carried out over a period of two years from 2006 – 2008 at five sentinel hospitals in the cities of Karachi, Lahore, Rawalpindi, and Peshawar. Stool samples collected from children under five years of age hospitalized with severe acute watery diarrhea were tested for rotavirus antigen via enzyme immunoassay (EIA) (IDEA REF K6020 Oxoid Ltd (Ely), Cambridge, United Kingdom). A subset of EIA positive stool samples were further processed for genotyping.

Results

6679 children were enrolled and stool specimens of 2039 (30.5%) were positive for rotavirus. Rotavirus positivity ranged from 16.3% to 39.4% in the 5 hospitals with highest positivity in Lahore. 1241 (61%) of all rotavirus cases were in infants under one year of age. Among the strains examined for G-serotypes, the occurrence of G1, G2, G9 and G4 strains was found to be 28%, 24%, 14% and 13%, respectively. Among P-types, the most commonly occurring strains were P6 (31.5%) followed by P8 (20%) and P4 (12%). Prevalent rotavirus genotype in hospitalized children of severe diarrhea were G1P[8] 11.6% (69/593), followed by G2P[4] 10.4% (62/593), and G4P[6] 10.1% (60/593).

Conclusions

Approximately one third of children hospitalized with severe gastroenteritis in urban centers in Pakistan have rotavirus. Introduction of rotavirus vaccine in Pakistan''s national immunization program could prevent many severe episodes and diarrheal deaths.     

Use of response surface methodology to study the effect of media composition on aflatoxin production by Aspergillus flavus

Aflatoxins are one of the most important secondary metabolites. These extrolites are produced by a number of Aspergillus fungi. In this study, we demonstrate the effect of media components and enhanced aflatoxin yield shown by A. flavus using response surface methodology in response to different nutrients. Different components of a chemically defined media that influence the aflatoxin production were monitored using Plackett–Burman experimental design and further optimized by Box–Behnken factorial design of response surface methodology in liquid culture. Interactions were studied with five variables, namely sorbitol, fructose, ammonium sulfate, KH₂PO₄, and MgSO₄.7H₂O. Maximum aflatoxin production was envisaged in medium containing 4.94 g/l sorbitol, 5.56 g/l fructose, 0.62 g/l ammonium sulfate, 1.33 g/l KH₂PO₄, and 0.65 g/l MgSO₄·7H₂O using response surface plots and the point prediction tool of the DESIGN EXPERT 8.1.0 (Stat-Ease, USA) software. However, a production of 5.25 μg/ml aflatoxin production was obtained, which was in agreement with the prediction observed in verification experiment. The other component (MgSO₄.7H₂O) was found to be an insignificant variable.     

Induction of phenoloxidase and other immunological activities in Sydney rock oysters challenged with microbial pathogen-associate molecular patterns

This study investigates the effects of two pathogen-associated molecular patterns (PAMPs), LPS and zymosan, on the Sydney rock oyster (Saccostrea glomerata) immune system. Phenoloxidase and phagocytic activities, total and differential haemocyte frequencies, as well as peroxide and superoxide concentrations were measured after the injection of lipopolysaccharide and zymosan. All of the immunological parameters were induced by both PAMPs. Phenoloxidase (monophenolase and diphenolase) and phagocytic activities, as well as the frequencies of phenoloxidase-positive haemocytes, hyalinocytes and granulocytes in the haemolymph, increased within 24 h of PAMP injection. Values for all of these parameters peaked within 48 h of challenge and began to decrease to levels that were indistinguishable from those of controls within 96h. The only exception to this pattern was diphenolase activity, which remained elevated for at least 96 h. Control saline injections that lacked PAMPs also induced responses in most of the parameters measured. However, reactions to saline injections were of far lower magnitude compared to those induced by PAMPs. All of the data suggest that the phenoloxidase and phagocytic systems of oysters are inducible components of the Sydney rock oyster immune system, and that induction is primarily due to increased frequencies of specialised haemocytes in the haemolymph.