Phosphorylation decelerates conformational dynamics in bacterial translation elongation factors

Bacterial protein synthesis is intricately connected to metabolic rate. One of the ways in which bacteria respond to environmental stress is through posttranslational modifications of translation factors. Translation elongation factor Tu (EF-Tu) is methylated and phosphorylated in response to nutrient starvation upon entering stationary phase, and its phosphorylation is a crucial step in the pathway toward sporulation. We analyze how phosphorylation leads to inactivation of Escherichia coli EF-Tu. We provide structural and biophysical evidence that phosphorylation of EF-Tu at T382 acts as an efficient switch that turns off protein synthesis by decoupling nucleotide binding from the EF-Tu conformational cycle. Direct modifications of the EF-Tu switch I region or modifications in other regions stabilizing the β-hairpin state of switch I result in an effective allosteric trap that restricts the normal dynamics of EF-Tu and enables the evasion of the control exerted by nucleotides on G proteins. These results highlight stabilization of a phosphorylation-induced conformational trap as an essential mechanism for phosphoregulation of bacterial translation and metabolism. We propose that this mechanism may lead to the multisite phosphorylation state observed during dormancy and stationary phase

The effect of infectious dose on humoral and cellular immune responses in Chlamydophila caviae primary ocular infection

Following infection, the balance between protective immunity and immunopathology often depends on the initial infectious load. Several studies have investigated the effect of infectious dose; however, the mechanism by which infectious dose affects disease outcomes and the development of a protective immune response is not known. The aim of this study was to investigate how the infectious dose modulates the local and systemic humoral and the cellular immune responses during primary ocular chlamydial infection in the guinea pig animal model. Guinea pigs were infected by ocular instillation of a Chlamydophila caviae-containing eye solution in the conjunctival sac in three different doses: 1x10(2), 1x10(4), and 1x10(6) inclusion forming units (IFUs). Ocular pathology, chlamydial clearance, local and systemic C. caviae-specific humoral and cellular immune responses were assessed. All inocula of C. caviae significantly enhanced the local production of C. caviae-specific IgA in tears, but only guinea pigs infected with the higher doses showed significant changes in C. caviae-specific IgA levels in vaginal washes and serum. On complete resolution of infection, the low dose of C. caviae did not alter the ratio of CD4(+) and CD8(+) cells within guinea pigs' submandibular lymph node (SMLN) lymphocytes while the higher doses increased the percentages of CD4(+) and CD8(+) cells within the SMLN lymphocytes. A significant negative correlation between pathology intensity and the percentage of CD4(+) and CD8(+) cells within SMLN lymphocyte pool at selected time points post-infection was recorded for both 1x10(4), and 1x10(6) IFU infected guinea pigs. The relevance of the observed dose-dependent differences on the immune response should be further investigated in repeated ocular chlamydial infections

Evaluation of five immunoassays for detection of Chlamydia psittaci in cloacal and conjunctival specimens from turkeys9199

Five commercially available immunoassays were evaluated for the detection of Chlamydia psittaci in cloacal and conjunctival swabs from industrially raised turkeys: IMAGEN (DAKO Diagnostics, Ely, Cambridgeshire, United Kingdom), Chlamydia CEL-VET IF (Cellabs, Brookvale, Australia), IDEIA (DAKO Diagnostics), CELISA (Cellabs), and CLEARVIEW (Unipath, Bedford, United Kingdom). Results were compared with isolation in Buffalo Green Monkey cells as a reference method. For the conjunctival samples, the sensitivities of the IMAGEN test, the Chlamydia CEL-VET IF test, the IDEIA, the CELISA, and the CLEARVIEW test were found to be 100, 66, 0, 0, and 0%, respectively, as compared to the reference test. Also for the conjunctival samples, the specificities of the IMAGEN test, the Chlamydia CEL-VET IF test, and the IDEIA were found to be 100, 11, and 92.8%, respectively. For the cloacal specimens, the sensitivities of the IMAGEN test, the Chlamydia CEL-VET IF test, the IDEIA, the CELISA, and the CLEARVIEW test were found to be 100, 93.3, 26.6, 0, and 53.3%, respectively. Also for the cloacal specimens, the specificities of the IMAGEN test, the Chlamydia CEL-VET IF test, the IDEIA, and the CLEARVIEW test were found to be 92, 12, 100, and 88%, respectively. The IMAGEN test was the most sensitive and specific direct chlamydia antigen detection test for cloacal and conjunctival samples from turkeys</p

Evaluation of five immunoassays for detection of Chlamydia psittaci in cloacal and conjunctival specimens from turkeys.

Five commercially available immunoassays were evaluated for the detection of Chlamydia psittaci in cloacal and conjunctival swabs from industrially raised turkeys: IMAGEN (DAKO Diagnostics, Ely, Cambridgeshire, United Kingdom), Chlamydia CEL-VET IF (Cellabs, Brookvale, Australia), IDEIA (DAKO Diagnostics), CELISA (Cellabs), and CLEARVIEW (Unipath, Bedford, United Kingdom). Results were compared with isolation in Buffalo Green Monkey cells as a reference method. For the conjunctival samples, the sensitivities of the IMAGEN test, the Chlamydia CEL-VET IF test, the IDEIA, the CELISA, and the CLEARVIEW test were found to be 100, 66, 0, 0, and 0%, respectively, as compared to the reference test. Also for the conjunctival samples, the specificities of the IMAGEN test, the Chlamydia CEL-VET IF test, and the IDEIA were found to be 100, 11, and 92.8%, respectively. For the cloacal specimens, the sensitivities of the IMAGEN test, the Chlamydia CEL-VET IF test, the IDEIA, the CELISA, and the CLEARVIEW test were found to be 100, 93.3, 26.6, 0, and 53.3%, respectively. Also for the cloacal specimens, the specificities of the IMAGEN test, the Chlamydia CEL-VET IF test, the IDEIA, and the CLEARVIEW test were found to be 92, 12, 100, and 88%, respectively. The IMAGEN test was the most sensitive and specific direct chlamydia antigen detection test for cloacal and conjunctival samples from turkeys

The prevalence of Chlamydia psittaci infections in Belgian commercial turkey poults7197

The prevalence of Chlamydia psittaci infections in Belgian commercial turkey poults was examined and a follow-up study of one Belgian turkey flock was performed. Sera were examined for the presence of anti-chlamydia antibodies by immunoblotting. Cloacal and conjunctival swab smears and lung impression smears were examined for the presence of chlamydial antigen using the IMAGEN Chlamydia immunofluorescence test. Anti-chlamydia antibodies were found in 90 of 100 sera collected at slaughter from turkeys raised during the summer of 1992. The following winter, 73 of 100 sera reacted positively. On all twenty farms examined during 1992, turkeys were positive for anti-chlamydial antibodies. During 1993, chlamydial antigen was detected in swabs from 20 of 40 slaughterhouse turkeys tested. Antigen was found more often in the cloaca than in the conjunctiva. Chlamydial antigen was detected in samples from each of the 4 farms examined. The follow-up study on a turkey farm, sampling the birds at weekly intervals from one week old until 12 weeks of age, revealed that chlamydial antigen and anti-chlamydial antibodies were present during the whole period. During 1994, chlamydial antigen was detected in 45 of 60 lungs from slaughterhouse turkeys from all of 6 farms. During 1995, chlamydial antigen was detected in 41 of 54 lungs of 6 week old commercial turkey poults. The results of the present study indicate that Chlamydia psittaci infections are highly prevalent amongst Belgian commercial turkey poults with apparently little seasonal or year-to-year variation and that turkeys can contract the infection at an early age</p

The prevalence of Chlamydia psittaci infections in Belgian commercial turkey poults

The prevalence of Chlamydia psittaci infections in Belgian commercial turkey poults was examined and a follow-up study of one Belgian turkey flock was performed. Sera were examined for the presence of anti-chlamydia antibodies by immunoblotting. Cloacal and conjunctival swab smears and lung impression smears were examined for the presence of chlamydial antigen using the IMAGEN Chlamydia immunofluorescence test. Anti-chlamydia antibodies were found in 90 of 100 sera collected at slaughter from turkeys raised during the summer of 1992. The following winter, 73 of 100 sera reacted positively. On all twenty farms examined during 1992, turkeys were positive for anti-chlamydial antibodies. During 1993, chlamydial antigen was detected in swabs from 20 of 40 slaughterhouse turkeys tested. Antigen was found more often in the cloaca than in the conjunctiva. Chlamydial antigen was detected in samples from each of the 4 farms examined. The follow-up study on a turkey farm, sampling the birds at weekly intervals from one week old until 12 weeks of age, revealed that chlamydial antigen and anti-chlamydial antibodies were present during the whole period. During 1994, chlamydial antigen was detected in 45 of 60 lungs from slaughterhouse turkeys from all of 6 farms. During 1995, chlamydial antigen was detected in 41 of 54 lungs of 6 week old commercial turkey poults. The results of the present study indicate that Chlamydia psittaci infections are highly prevalent amongst Belgian commercial turkey poults with apparently little seasonal or year-to-year variation and that turkeys can contract the infection at an early age

Optimization of polymerase chain reaction for detection of Clostridium botulinum type C and D in bovine samples

Botulism is a rare but serious paralytic illness caused by a nerve toxin that is produced by the bacterium Clostridium botulinum. The economic, medical and alimentary consequences can be catastrophic in case of an epizooty. A polymerase chain reaction (PCR)-based assay was developed for the detection of C. botulinum toxigenic strains type C and D in bovine samples. This assay has proved to be less expensive, faster and simpler to use than the mouse bioassay, the current reference method for diagnosis of C. botulinum toxigenic strains. Three pairs of primers were designed, one for global detection of C. botulinum types C and D (primer pair Y), and two strain-specific pairs specifically designed for types C (primer pair VC) and D (primer pair VD). The PCR amplification conditions were optimized and evaluated on 13 bovine and two duck samples that had been previously tested by the mouse bioassay. In order to assess the impact of sample treatment, both DNA extracted from crude samples and three different enrichment broths (TYG, CMM, CMM followed by TYG) were tested. A 100% sensitivity was observed when samples were enriched for 5 days in CMM followed by 1 day in TYG broth. False-negative results were encountered when C. botulinum was screened for in crude samples. These findings indicate that the current PCR is a reliable method for the detection of C. botulinum toxigenic strains type C and D in bovine samples but only after proper enrichment in CMM and TYG broth