Characterization of the SecA2 Protein Export Pathway of Mycobacteria

Nearly one-third of the world's population is infected with Mycobacterium tuberculosis, the bacterium that causes tuberculosis. To establish and maintain infection, M. tuberculosis uses surface and secreted proteins to modulate the host immune response. There are several dedicated export machines that transport surface and secreted proteins from their site of synthesis in the cytoplasm across the bacterial cytoplasmic membrane. The bulk of protein export across the cytoplasmic membrane is carried out by the Sec pathway. Energy for Sec-dependent protein export is provided by the essential ATPase SecA. Recently, a small subset of Gram positive bacteria and mycobacteria were found to possess two SecA homologs, SecA1 and SecA2. In M. tuberculosis and the non-pathogenic model mycobacterium M. smegmatis, SecA1 is essential for general protein export and is the presumed "housekeeping" SecA, while SecA2 is an accessory SecA specific for a subset of exported proteins. In this work, we describe our initial attempts to characterize the mechanism behind SecA2-mediated protein export in mycobacteria. We began by comparing similarities and differences between SecA1 and SecA2. One striking difference we discovered was that SecA1 and SecA2 localize to different cellular compartments. By comparing a structural prediction of SecA2 to the published crystal structure of SecA1, we identified putative structural differences between the two SecAs. Both SecA1 and SecA2 have predicted ATP binding sites. We showed that M. tuberculosis SecA1 and SecA2 bind and hydrolyze ATP in vitro. By constructing a secA2 mutant that encodes a protein defective in ATP binding, we also demonstrated that ATP binding is required for normal SecA2 function in vivo. Upon subsequent analysis, we found that SecA2 mutants unable to bind ATP were dominant negative. We used this dominant negative phenotype as a tool to study SecA2 by performing a suppressor screen. We isolated intragenic suppressors and used them to identify structural subdomains of SecA2 that are important for function. While we have not yet identified the extragenic suppressors, we believe they represent a powerful tool for identifying SecA2-interacting proteins. Identifying SecA2-interacting proteins will be crucial to fully understand the accessory SecA2 protein export pathway of mycobacteria. Finally, we show for the first time in any bacterium with two SecA proteins that the canonical SecA1 protein is also required to export SecA2-dependent substrates. By developing a better understanding of the SecA2 pathway, we hope to increase our knowledge of mycobacterial physiology to enable the design of new anti-tuberculosis therapies

A new twist on an old pathway – accessory secretion systems

The export of proteins from their site of synthesis in the cytoplasm across the inner membrane is an important aspect of bacterial physiology. Because the location of extracytoplasmic proteins is ideal for host-pathogen interactions, protein export is also important to bacterial virulence. In bacteria there are conserved protein export systems that are responsible for the majority of protein export: the general secretion (Sec) pathway and the twin-arginine translocation (Tat) pathway. In some bacteria, there are also specialized export systems dedicated to exporting specific subsets of proteins. In this review, we discuss a specialized export system that exists in some Gram-positive bacteria and mycobacteria – the accessory Sec system. The common element to the accessory Sec system is an accessory SecA protein called SecA2. Here we present our current understanding of accessory Sec systems in Streptococcus gordonii, Streptococcus parasanguinis, Mycobacterium smegmatis, Mycobacterium tuberculosis, and Listeria monocytogenes, making an effort to highlight apparent similarities and differences between the systems. We also review the data showing that accessory Sec systems can contribute to bacterial virulence

Validation of a 40-Gene Expression Profile Test to Predict Metastatic Risk in Localized High-Risk Cutaneous Squamous Cell Carcinoma

Background: Current staging systems for cutaneous squamous cell carcinoma (cSCC) have limited positive predictive value (PPV) for identifying patients who will experience metastasis. Objective: To develop and validate a gene expression profile (GEP) test for predicting risk for metastasis in localized, high-risk cSCC with the goal of improving risk-directed patient management. Methods: Archival formalin-fixed paraffin-embedded primary cSCC tissue and clinicopathologic data (n=586) were collected from 23 independent centers in a prospectively designed study. A GEP signature was developed using a discovery cohort (n=202) and validated in a separate, non-overlaping, independent cohort (n=324). Results: A prognostic, 40-gene expression profile (40-GEP) test was developed and validated, stratifying high-risk cSCC patients into classes based on metastasis risk: Class 1 (low-risk), Class 2A (high-risk), and Class 2B (highest-risk). For the validation cohort, 3-year metastasis-free survival (MFS) rates were 91.4%, 80.6%, and 44.0%, respectively. A PPV of 60% was achieved for the highest-risk group (Class 2B), an improvement over staging systems; while negative predictive value, sensitivity, and specificity were comparable to staging systems. Limitations: Potential understaging of cases could affect metastasis rate accuracy.Conclusion: The 40-GEP test is an independent predictor of metastatic risk that can complement current staging systems for patients with high-risk cSCC

<i>Acinetobacter baumannii</i> Strains Deficient in the Clp Chaperone-Protease Genes Have Reduced Virulence in a Murine Model of Pneumonia

Acinetobacter baumannii has emerged as a significant opportunistic Gram-negative pathogen and causative agent of nosocomial pneumonia especially in immunocompromised individuals in intensive care units. Recent advances to understand the contribution and function of A. baumannii virulence factors in its pathogenesis have begun to elucidate how this bacterium interacts with immune cells and its interesting mechanisms for multi-antibiotic resistance. Taking advantage of the availability of the A. baumannii AB5075 transposon mutant library, we investigated the impact of the A. baumannii Clp genes, which encode for a chaperone-protease responsible for the degradation of misfolded proteins, on bacterial virulence in a model of pneumonia using C57BL/6 mice and survival within J774.16 macrophage-like cells. Clp-protease A. baumannii mutants exhibit decreased virulence in rodents, high phagocytic cell-mediated killing and reduced biofilm formation. Capsular staining showed evidence of encapsulation in A. baumannii AB5075 and Clp-mutant strains. Surprisingly, clpA and clpS mutants displayed irregular cell morphology, which may be important in the biofilm structural deficiencies observed in these strains. Interestingly, clpA showed apical-like growth, proliferation normally observed in filamentous fungi. These findings provide new information regarding A. baumannii pathogenesis and may be important for the development of therapies intended at reducing morbidity and mortality associated with this remarkable pathogen

Identification of Functional Tat Signal Sequences in Mycobacterium tuberculosis Proteins▿ †

The twin-arginine translocation (Tat) pathway is a system used by some bacteria to export proteins out from the cytosol to the cell surface or extracellular environment. A functional Tat pathway exists in the important human pathogen Mycobacterium tuberculosis. Identification of the substrates exported by the Tat pathway can help define the role that this pathway plays in the physiology and pathogenesis of M. tuberculosis. Here we used a reporter of Tat export, a truncated β-lactamase, ′BlaC, to experimentally identify M. tuberculosis proteins with functional Tat signal sequences. Of the 13 proteins identified, one lacks the hallmark of a Tat-exported substrate, the twin-arginine dipeptide, and another is not predicted by in silico analysis of the annotated M. tuberculosis genome. Full-length versions of a subset of these proteins were tested to determine if the native proteins are Tat exported. For three proteins, expression in a Δtat mutant of Mycobacterium smegmatis revealed a defect in precursor processing compared to expression in the wild type, indicating Tat export of the full-length proteins. Conversely, two proteins showed no obvious Tat export in M. smegmatis. One of this latter group of proteins was the M. tuberculosis virulence factor phospholipase C (PlcB). Importantly, when tested in M. tuberculosis a different result was obtained and PlcB was exported in a twin-arginine-dependent manner. This suggests the existence of an M. tuberculosis-specific factor(s) for Tat export of a proven virulence protein. It also emphasizes the importance of domains beyond the Tat signal sequence and bacterium-specific factors in determining if a given protein is Tat exported

ATPase Activity of Mycobacterium tuberculosis SecA1 and SecA2 Proteins and Its Importance for SecA2 Function in Macrophages▿

The Sec-dependent translocation pathway that involves the essential SecA protein and the membrane-bound SecYEG translocon is used to export many proteins across the cytoplasmic membrane. Recently, several pathogenic bacteria, including Mycobacterium tuberculosis, were shown to possess two SecA homologs, SecA1 and SecA2. SecA1 is essential for general protein export. SecA2 is specific for a subset of exported proteins and is important for M. tuberculosis virulence. The enzymatic activities of two SecA proteins from the same microorganism have not been defined for any bacteria. Here, M. tuberculosis SecA1 and SecA2 are shown to bind ATP with high affinity, though the affinity of SecA1 for ATP is weaker than that of SecA2 or Escherichia coli SecA. Amino acid substitution of arginine or alanine for the conserved lysine in the Walker A motif of SecA2 eliminated ATP binding. We used the SecA2(K115R) variant to show that ATP binding was necessary for the SecA2 function of promoting intracellular growth of M. tuberculosis in macrophages. These results are the first to show the importance of ATPase activity in the function of accessory SecA2 proteins

The Accessory SecA2 System of Mycobacteria Requires ATP Binding and the Canonical SecA1*S⃞

In bacteria, the majority of exported proteins are transported by the general Sec pathway from their site of synthesis in the cytoplasm across the cytoplasmic membrane. The essential SecA ATPase powers this Sec-mediated export. Mycobacteria possess two nonredundant SecA homologs: SecA1 and SecA2. In pathogenic Mycobacterium tuberculosis and the nonpathogenic model mycobacterium Mycobacterium smegmatis, SecA1 is essential for protein export and is the “housekeeping” SecA, whereas SecA2 is an accessory SecA that exports a specific subset of proteins. In M. tuberculosis the accessory SecA2 pathway plays a role in virulence. In this study, we uncovered basic properties of the mycobacterial SecA2 protein and its pathway for exporting select proteins. By constructing secA2 mutant alleles that encode proteins defective in ATP binding, we showed that ATP binding is required for SecA2 function. SecA2 mutant proteins unable to bind ATP were nonfunctional and dominant negative. By evaluating the subcellular distribution of each SecA, SecA1 was shown to be equally divided between cytosolic and cell envelope fractions, whereas SecA2 was predominantly localized to the cytosol. Finally, we showed that the canonical SecA1 has a role in the process of SecA2-dependent export. The accessory SecA2 export system is important to the physiology and virulence of mycobacteria. These studies help establish the mechanism of this new type of specialized protein export pathway

Making a beta-barrel: assembly of outer membrane proteins in Gram-negative bacteria

The outer membrane (OM) of Gram negative bacteria is an essential organelle that serves as a selective permeability barrier by keeping toxic compounds out of the cell while allowing vital nutrients in. How the OM and its constituent lipid and protein components are assembled remains an area of active research. In this review, we describe our current understanding of how outer membrane proteins (OMPs) are delivered to and then assembled in the OM of the model Gram-negative organism Escherichia coli