Characterizing the Role of CpsA in Mycobacterial Pathogenesis

Mycobacterium tuberculosis infects one-third of the world’s population and causes an estimated 2 million deaths per year, more than any other single bacterial pathogen. The inadequacies of existing tuberculosis therapies demand the discovery of novel agents to treat M. tuberculosis infection, which requires mechanistic insight into the pathways involved in mycobacterial pathogenesis. We identified an unanticipated role of the M. tuberculosis protein CpsA in preventing phago-lysosome fusion by host macrophages, resulting in enhanced intracellular survival of bacteria within the host. Strains of M. tuberculosis lacking cpsA are severely attenuated in both macrophage and mouse models, thus establishing CpsA as a crucial virulence factor in M. tuberculosis infection. CpsA is a yet uncharacterized secreted protein, belonging to the LytR–CpsA–PsR family, that we found interacts with host proteins NDP52 and TAX1BP1, which are involved in intracellular trafficking. Here, we sought to (1) examine whether CpsA is sufficient to confer virulence to Mycobacterium smegmatis, a rapidly-growing, non-pathogenic bacterium that does not contain CpsA naturally, and (2) determine which domains of CpsA are required for interacting with host proteins and contributing to virulence. We infected RAW264.7 macrophages with M. smegmatis expressing M. tuberculosis cpsA and vector control and scored for intracellular survival of bacteria. Our preliminary data suggest that CpsA confers enhanced intracellular survival to M. smegmatis, as compared to vector control. To characterize CpsA interaction with TAX1BP1 and NDP52, we generated deletion constructs of CpsA and, using a yeast two-hybrid system, investigated which domains of the CpsA protein are required for the interaction with these host proteins. Follow up work involves determining which domains are also required for virulence. Overall, we hope that by studying the molecular mechanisms by which M. tuberculosis CpsA sabotages cellular functions, we can better understand both the host immunity and mycobacterial pathogenesis

A Hybrid Analysis for Security Protocols with State

Cryptographic protocols rely on message-passing to coordinate activity among principals. Each principal maintains local state in individual local sessions only as needed to complete that session. However, in some protocols a principal also uses state to coordinate its different local sessions. Sometimes the non-local, mutable state is used as a means, for example with smart cards or Trusted Platform Modules. Sometimes it is the purpose of running the protocol, for example in commercial transactions. Many richly developed tools and techniques, based on well-understood foundations, are available for design and analysis of pure message-passing protocols. But the presence of cross-session state poses difficulties for these techniques. In this paper we provide a framework for modeling stateful protocols. We define a hybrid analysis method. It leverages theorem-proving---in this instance, the PVS prover---for reasoning about computations over state. It combines that with an "enrich-by-need" approach---embodied by CPSA---that focuses on the message-passing part. As a case study we give a full analysis of the Envelope Protocol, due to Mark Ryan

Security Theorems via Model Theory

A model-theoretic approach can establish security theorems for cryptographic protocols. Formulas expressing authentication and non-disclosure properties of protocols have a special form. They are quantified implications for all xs . (phi implies for some ys . psi). Models (interpretations) for these formulas are *skeletons*, partially ordered structures consisting of a number of local protocol behaviors. Realized skeletons contain enough local sessions to explain all the behavior, when combined with some possible adversary behaviors. We show two results. (1) If phi is the antecedent of a security goal, then there is a skeleton A_phi such that, for every skeleton B, phi is satisfied in B iff there is a homomorphism from A_phi to B. (2) A protocol enforces for all xs . (phi implies for some ys . psi) iff every realized homomorphic image of A_phi satisfies psi. Hence, to verify a security goal, one can use the Cryptographic Protocol Shapes Analyzer CPSA (TACAS, 2007) to identify minimal realized skeletons, or "shapes," that are homomorphic images of A_phi. If psi holds in each of these shapes, then the goal holds

Phylogenetic distribution and membrane topology of the LytR-CpsA-Psr protein family

BACKGROUND: The bacterial cell wall is the target of many antibiotics and cell envelope constituents are critical to host-pathogen interactions. To combat resistance development and virulence, a detailed knowledge of the individual factors involved is essential. Members of the LytR-CpsA-Psr family of cell envelope-associated attenuators are relevant for beta-lactam resistance, biofilm formation, and stress tolerance, and they are suggested to play a role in cell wall maintenance. However, their precise function is still unknown. This study addresses the occurrence as well as sequence-based characteristics of the LytR-CpsA-Psr proteins. RESULTS: A comprehensive list of LytR-CpsA-Psr proteins was established, and their phylogenetic distribution and clustering into subgroups was determined. LytR-CpsA-Psr proteins were present in all Gram-positive organisms, except for the cell wall-deficient Mollicutes and one strain of the Clostridiales. In contrast, the majority of Gram-negatives did not contain LytR-CpsA-Psr family members. Despite high sequence divergence, the LytR-CpsA-Psr domains of different subclusters shared a highly similar, predicted mixed alpha/beta-structure, and conserved charged residues. PhoA fusion experiments, using MsrR of Staphylococcus aureus, confirmed membrane topology predictions and extracellular location of its LytR-CpsA-Psr domain. CONCLUSIONS: The LytR-CpsA-Psr domain is unique to bacteria. The presence of diverse subgroups within the LytR-CpsA-Psr family might indicate functional differences, and could explain variations in phenotypes of respective mutants reported. The identified conserved structural elements and amino acids are likely to be important for the function of the domain and will help to guide future studies of the LytR-CpsA-Psr proteins

Analysis of the streptococcal cpsa protein in dna-binding and regulation of capsule and cell wall maintenance

The systemic pathogens Streptococcus agalactiae (GBS) and Streptococcus pneumoniae remain a significant threat to human health worldwide. The ability of these organisms to cause systemic disease is compounded by the production of a polysaccharide capsule that provides immune evasion function. The production of the polysaccharide capsule in systemic streptococcal pathogens is controlled in part by the membrane bound protein CpsA. These studies analyze the contribution of CpsA to regulation of capsule level in the model aquatic pathogen Streptococcus iniae and human specific pathogen GBS, and how this regulation affects virulence in in-vitro, ex-vivo, and in-vivo models of pathogenesis. We have determined that the membrane topology of the CpsA protein consists of a small cytoplasmic N-terminus, and a large extracellular C-terminus that contains the conserved DNA_PPF and LytR protein domains. The cytoplasmic N-terminal region in its entirety is capable of binding specifically to the capsule operon cpsA promoter in S. iniae, and to two putative promoter elements in GBS which include the capsule operon cpsA promoter and the internal cpsE promoter. Additionally, CpsA is a modular protein, with the cytoplasmic N-terminus as a capsule-activating domain, the DNA_PPF region as a capsule-repressing domain, and the LytR region as a control domain that regulates the activities of the other two domains. CpsA also appears to regulate cell wall maintenance, as truncation or removal of CpsA results in longer chains of cocci in both S. iniae and S. agalactiae, a phenotype that is associated with altered antimicrobial resistance and autolysis activity in S. iniae. Taken together, CpsA contributes to the complex regulatory scheme controlling capsule and cell wall, the two major constituents of bacterial cell surface macromolecular structure, and does so in a way that influences pathogenesis during systemic disease. The insights gained through these studies indicates that CpsA can be targeted in a way that is detrimental to bacterial survival in the context of systemic disease, suggesting CpsA may be an important future target of anti-virulence antimicrobial therapy

Bolshevizing communist parties: The Algerian and South African experiences

In 1924 and 1925 the Comintern introduced its policy of Bolshevization. A goal of Bolshevization was the creation of mass-based communist parties. In settler societies this meant that the local communist party should aim to be demographically representative of the entire population. This article traces the efforts of the communist parties in Algeria and South Africa to indigenize, seeking to explain why their efforts had such diverse outcomes. It examines four variables: the patterns of working-class formation; the socialist tradition of each country; the relationship between the Comintern and the two communist parties; and the level of repression against communists in both societies. The cumulative weight of the variables in the Algerian case helps to explain why communist activity in the 1920s - including the communist party's ability to indigenize - was far more difficult in Algeria than South Africa