Mycobacterium marinum SecA2 promotes stable granulomas and induces tumor necrosis factor alpha in vivo.

SecA2 is an ATPase present in some pathogenic Gram-positive bacteria, is required for translocation of a limited set of proteins across the cytosolic membrane, and plays an important role in virulence in several bacteria, including mycobacteria that cause diseases such as tuberculosis and leprosy. However, the mechanisms by which SecA2 affects virulence are incompletely understood. To investigate whether SecA2 modulates host immune responses in vivo, we studied Mycobacterium marinum infection in two different hosts: an established zebrafish model and a recently described mouse model. Here we show that M. marinum ΔsecA2 was attenuated for virulence in both host species and SecA2 was needed for normal granuloma numbers and for optimal tumor necrosis factor alpha response in both zebrafish and mice. M. marinum ΔsecA2 was more sensitive to SDS and had unique protrusions from its cell envelope when examined by cryo-electron tomography, suggesting that SecA2 is important for bacterial cell wall integrity. These results provide evidence that SecA2 induces granulomas and is required for bacterial modulation of the host response because it affects the mycobacterial cell envelope.

EccA1, a component of the Mycobacterium marinum ESX-1 protein virulence factor secretion pathway, regulates mycolic acid lipid synthesis.

Pathogenic mycobacteria, which cause multiple diseases including tuberculosis, secrete factors essential for disease via the ESX-1 protein export system and are partially protected from host defenses by their lipid-rich cell envelopes. These pathogenic features of mycobacterial biology are believed to act independently of each other. Key ESX-1 components include three ATPases, and EccA1 (Mycobacterium marinum MMAR_5443; M. tuberculosis Rv3868) is the least characterized. Here we show that M. marinum EccA1's ATPase activity is required for ESX-1-mediated protein secretion, and surprisingly for the optimal synthesis of mycolic acids, integral cell-envelope lipids. Increased mycolic acid synthesis defects, observed when an EccA1-ATPase mutant is expressed in an eccA1-null strain, correlate with decreased in vivo virulence and intracellular growth. These data suggest that two mycobacterial virulence hallmarks, ESX-1-dependent protein secretion and mycolic acid synthesis, are critically linked via EccA1.

Outcome of optical penetrating keratoplasties at a tertiary care eye institute in Western India.

AIM: To study the indications, risk factors, postoperative course, and long-term survival of corneal transplants done for optical purposes. DESIGN: Retrospective case series. MATERIALS AND METHODS: Data were obtained by reviewing the records of 181 patients operated at our institute (H.V. Desai Eye Hospital) between October 2005 and October 2007 for optical penetrating keratoplasty. Patients with less than one year of follow up, pediatric cases, therapeutic, tectonic, and lamellar keratoplasties were excluded. Kaplan Meier survival analysis was used to calculate median survival time of grafts and to see correlation between nine variables viz. age, gender, corneal vascularization, previous failed grafts, previous Herpes Simplex keratitis, post-perforation corneal scars, donor tissue quality, graft size, type of surgery and follow-up. These variables were also used for univariate and multivariate analysis using Cox Proportional Hazard Regression Modeling. RESULTS: Median survival of the cohort was 27 months (95% confidence interval: 20.47-33.52). One- and two-year survival rates were 65% and 52.5%, respectively. Median survival was significantly lower in poor prognosis cases (14 months) than good prognosis cases (27 months, P = 0.0405). Graft survival was lower in vascularized corneas (18.55 months, P = 0.030) and in post-perforation corneal scars (17.96 months, P = 0.09, borderline significance). Multivariate analysis showed that the same factors were predictive of graft failure. CONCLUSION: Long-term survival of grafts at our center is different from centers in western world. More high-risk cases, paucity of excellent quality donor corneas, and differences in patient profile could be the contributory factors.

A delicate dance: host response to mycobacteria.

Mycobacterium tuberculosis is an enormously successful human pathogen that can infect its host for decades without causing clinical disease, only to reactivate when host immunity is compromised. A normal immune response thus contains bacterial spread without inducing sterilizing immunity, therefore benefitting both host and pathogen. Recent work has begun to outline the complexity of this host-pathogen interaction and to reveal how the homeostatic balance between the two is achieved. This review focuses on two significant aspects of this delicate dance: the host's initial innate response and the mature granuloma that later contains the pathogen. Here, we review the fine balance of inflammatory events triggered or controlled by both the host and bacteria and implications for the survival of each.

Safety and efficacy of phacoemulsification compared with manual small-incision cataract surgery by a randomized controlled clinical trial: six-week results.

OBJECTIVE: To compare the efficacy, safety, and refractive errors of astigmatism after cataract surgery by phacoemulsification and manual small-incision cataract surgery techniques. DESIGN: Masked randomized control clinical trial. PARTICIPANTS: Four hundred eyes of 400 patients, 1:1 randomization with half in each arm of the trial. METHODS: A total of 400 eyes was assigned randomly to either phacoemulsification or small-incision groups after informed consent and were operated on by 4 surgeons. They were masked to the technique of surgery before, during, and after cataract surgery and followed up to 1 year after surgery. The intraoperative and postoperative complications, uncorrected and best-corrected visual acuity, and astigmatism were recorded at 1 and 6 weeks postoperatively. MAIN OUTCOME MEASURES: The proportion of patients achieving visual acuity better than or equal to 6/18 with and without spectacles after cataract surgery in the operated eye up to 6 weeks, postoperative astigmatism, and complications during and after surgery. RESULTS: This article reports clinical outcomes up to 6 weeks. Three hundred eighty-three of 400 (95.75%) patients completed the 1-week follow-up, and 372 of 400 (93%) patients completed the 6-week follow-up. One hundred thirty-one of 192 (68.2%) patients in the phacoemulsification group and 117 of 191 (61.25%) patients in the small-incision group had uncorrected visual acuity better than or equal to 6/18 at 1 week (P = 0.153). One hundred fifty of 185 (81.08%) patients of the phacoemulsification group and 133 of 187 (71.1%) patients of the small-incision group (P = 0.038) were better than or equal to 6/18 at the 6-week follow-up for presenting visual activity. Visual acuity improved to > or = 6/18 with best correction in 182 of 185 patients (98.4%) and 184 of 187 (98.4%) patients (P = 0.549), respectively. Poor outcome (postoperative visual acuity < 6/60) was noted in 1 of 185 (0.5%) in the phacoemulsification group and none in the small-incision group. The mode of astigmatism was 0.5 diopters (D) for the phacoemulsification group and 1.5 D for the small-incision group, and the average astigmatism was 1.1 D and 1.2 D, respectively. There was an intra-surgeon variation in astigmatism. The phacoemulsification group had 7 posterior capsular rents compared with 12 in the small-incision group, but the phacoemulsification group had more corneal edema on the first postoperative day. CONCLUSIONS: Both the phacoemulsification and the small-incision techniques are safe and effective for visual rehabilitation of cataract patients, although phacoemulsification gives better uncorrected visual acuity in a larger proportion of patients at 6 weeks.

Sculpting the proteome with AAA(+) proteases and disassembly machines.

Machines of protein destruction-including energy-dependent proteases and disassembly chaperones of the AAA(+) ATPase family-function in all kingdoms of life to sculpt the cellular proteome, ensuring that unnecessary and dangerous proteins are eliminated and biological responses to environmental change are rapidly and properly regulated. Exciting progress has been made in understanding how AAA(+) machines recognize specific proteins as targets and then carry out ATP-dependent dismantling of the tertiary and/or quaternary structure of these molecules during the processes of protein degradation and the disassembly of macromolecular complexes.

Communication between ClpX and ClpP during substrate processing and degradation.

In the ClpXP compartmental protease, ring hexamers of the AAA(+) ClpX ATPase bind, denature and then translocate protein substrates into the degradation chamber of the double-ring ClpP(14) peptidase. A key question is the extent to which functional communication between ClpX and ClpP occurs and is regulated during substrate processing. Here, we show that ClpX-ClpP affinity varies with the protein-processing task of ClpX and with the catalytic engagement of the active sites of ClpP. Functional communication between symmetry-mismatched ClpXP rings depends on the ATPase activity of ClpX and seems to be transmitted through structural changes in its IGF loops, which contact ClpP. A conserved arginine in the sensor II helix of ClpX links the nucleotide state of ClpX to the binding of ClpP and protein substrates. A simple model explains the observed relationships between ATP binding, ATP hydrolysis and functional interactions between ClpX, protein substrates and ClpP.

C-terminal domain mutations in ClpX uncouple substrate binding from an engagement step required for unfolding.

ClpX mediates ATP-dependent denaturation of specific target proteins and disassembly of protein complexes. Like other AAA + family members, ClpX contains an alphabeta ATPase domain and an alpha-helical C-terminal domain. ClpX proteins with mutations in the C-terminal domain were constructed and screened for disassembly activity in vivo. Seven mutant enzymes with defective phenotypes were purified and characterized. Three of these proteins (L381K, D382K and Y385A) had low activity in disassembly or unfolding assays in vitro. In contrast to wild-type ClpX, substrate binding to these mutants inhibited ATP hydrolysis instead of increasing it. These mutants appear to be defective in a reaction step that engages bound substrate proteins and is required both for enhancement of ATP hydrolysis and for unfolding/disassembly. Some of these side chains form part of the interface between the C-terminal domain of one ClpX subunit and the ATPase domain of an adjacent subunit in the hexamer and appear to be required for communication between adjacent nucleotide binding sites.