Insights into the Chlamydial Niche: The Dynamic Roles of Inclusion Membrane (Inc) Proteins in Chlamydia trachomatis Development

Chlamydia trachomatis (Ctr) is the leading cause of bacterial sexually transmitted infections. Ctr, an obligate intracellular bacterium, develops within a membrane-bound vacuole called an inclusion. The inclusion membrane is modified by chlamydial inclusion membrane (Inc) proteins, the functions of which are poorly characterized. Bacterial two-hybrid analyses found some Incs (e.g., IncF) interacted with numerous Incs while others (e.g., IncA) did not. We hypothesize that some Incs organize the inclusion through Inc-Inc interactions whereas other Incs promote chlamydial-host interactions by binding eukaryotic proteins. To test our hypothesis, we implemented the ascorbate peroxidase proximity labeling system (APEX2), which labels proximal proteins with biotin in vivo. We transformed Ctr with inducible expression constructs containing incF-APEX2, incATM(transmembrane domain)-APEX2, incA-APEX2, and APEX2 alone. Affinity purification-mass spectrometry (AP-MS) of biotinylated proteins from chlamydial infected monolayers, followed by Significance Analysis of INTeractome (SAINT), was used to identify significant proteins that are proximal to the Inc-APEX2 constructs from five biological replicates. Consistent with our hypothesis, IncF-APEX2 biotinylated more chlamydial Inc proteins, whereas IncA-APEX2 biotinylated more unique eukaryotic proteins. We validated a SAINT significant eukaryotic protein, LRRF1, at the inclusion membrane by immunofluorescence and determined that the Inc CT226 interacts with LRRF1. Next, we compared our datasets with other AP-MS inclusion studies, finding only seven proteins that were similarly identified, likely a reflection of the different experimental approaches. Importantly, for the first time, we were able to directly compare two Inc-APEX2 studies, which also revealed fewer similarly identified proteins than expected. This led us to hypothesize that the overexpression of certain Incs may alter the organization of the inclusion membrane. Previously, we described defects in inclusion expansion and the production of progeny when IncF-APEX2 was expressed at high levels from transformed Ctr. To further investigate the role of Incs in chlamydial development, we transformed Ctr with inducible expression plasmids containing FLAG-tagged incF, ct813, and ct226. The expression of IncF and CT813, but not CT226, altered inclusion development, as observed by smaller inclusions and decreased IncE intensity in the inclusion membrane. These data suggest that coordinated Inc expression and insertion into the inclusion membrane is essential for optimal inclusion development

Determinants of short and long term functional recovery after hospitalization for community-acquired pneumonia in the elderly: role of inflammatory markers

BACKGROUND: Hospitalization for older patients with community-acquired pneumonia (CAP) is associated with functional decline. Little is know about the relationship between inflammatory markers and determinants of functional status in this population. The aim of the study is to investigate the association between tumor necrosis factor (TNF)-α, C-reactive protein (CRP) and Activities of Daily Living, and to identify risk factors associated with one year mortality or hospital readmission. METHODS: 301 consecutive patients hospitalized for CAP (mean age 73.9 ± 5.3 years) in a University affiliated hospital over 18 month period were included. All patients were evaluated on admission to identify baseline demographic, microbiological, cognitive and functional characteristics. Serum levels for TNF-α and CRP were collected at the same time. Reassessment of functional status at discharge, and monthly thereafter till 3 months post discharge was obtained and compared with preadmission level to document loss or recovery of functionality. Outcome was assessed by the composite endpoint of hospital readmission or death from any cause up to one year post hospital discharge. RESULTS: 36% of patients developed functional decline at discharge and 11% had persistent functional impairment at 3 months. Serum TNF-α (odds ratio [OR] 1.12, 95% CI 1.08–1.15; p < 0.001) and the Charlson Index (OR = 1.39, 95% CI 1.14 to 1.71; p = 0.001) but not age, CRP, or cognitive status were independently associated with loss of functionality at the time of hospital discharge. Lack of recovery in functional status at 3 months was associated with impaired cognitive ability and preadmission comorbidities. In Cox regression analysis, persistent functional impairment at 3 months, impaired cognitive function, and the Charlson Index were highly predictive of one year hospital readmission or death. CONCLUSION: Serum TNF-α levels can be useful in determining patients at risk for functional impairment following hospitalization from CAP. Old patients with impaired cognitive function and preexisting comorbidities who exhibit delay in functional recovery at 3 months post discharge may be at high risk for hospital readmission and death. With the scarcity of resources, a future risk stratification system based on these findings might be proven helpful to target older patients who are likely to benefit from interventional strategies

The Complete Genome of Propionibacterium freudenreichii CIRM-BIA1T, a Hardy Actinobacterium with Food and Probiotic Applications

Background: Propionibacterium freudenreichii is essential as a ripening culture in Swiss-type cheeses and is also considered for its probiotic use [1]. This species exhibits slow growth, low nutritional requirements, and hardiness in many habitats. It belongs to the taxonomic group of dairy propionibacteria, in contrast to the cutaneous species P. acnes. The genome of the type strain, P. freudenreichii subsp. shermanii CIRM-BIA1 (CIP 103027T), was sequenced with an 11-fold coverage. Methodology/Principal Findings: The circular chromosome of 2.7 Mb of the CIRM-BIA1 strain has a GC-content of 67% and contains 22 different insertion sequences (3.5% of the genome in base pairs). Using a proteomic approach, 490 of the 2439 predicted proteins were confirmed. The annotation revealed the genetic basis for the hardiness of P. freudenreichii, as the bacterium possesses a complete enzymatic arsenal for de novo biosynthesis of aminoacids and vitamins (except panthotenate and biotin) as well as sequences involved in metabolism of various carbon sources, immunity against phages, duplicated chaperone genes and, interestingly, genes involved in the management of polyphosphate, glycogen and trehalose storage. The complete biosynthesis pathway for a bifidogenic compound is described, as well as a high number of surface proteins involved in interactions with the host and present in other probiotic bacteria. By comparative genomics, no pathogenicity factors found in P. acnes or in other pathogenic microbial species were identified in P. freudenreichii, which is consistent with the Generally Recognized As Safe and Qualified Presumption of Safety status of P. freudenreichii. Various pathways for formation of cheese flavor compounds were identified: the Wood-Werkman cycle for propionic acid formation, amino acid degradation pathways resulting in the formation of volatile branched chain fatty acids, and esterases involved in the formation of free fatty acids and esters. Conclusions/Significance: With the exception of its ability to degrade lactose, P. freudenreichii seems poorly adapted to dairy niches. This genome annotation opens up new prospects for the understanding of the P. freudenreichii probiotic activity

Risk and safety requirements for diagnostic and therapeutic procedures in allergology : World Allergy Organization Statement

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