Crystal structure of a variable region segment of Leptospira host-interacting outer surface protein, LigA, reveals the orientation of Ig-like domains.

Leptospiral immunoglobulin-like (Lig) protein family is a surface-exposed protein from the pathogenic Leptospira. The Lig protein family has been identified as an essential virulence factor of L. interrogan. One of the family members, LigA, contains 13 homologous tandem repeats of bacterial Ig-like (Big) domains in its extracellular portion. It is crucial in binding with the host's Extracellular matrices (ECM) and complement factors. However, its vital role in the invasion and evasion of pathogenic Leptospira, structural details, and domain organization of the extracellular portion of this protein are not explored thoroughly. Here, we described the first high-resolution crystal structure of a variable region segment (LigA8-9) of LigA at 1.87 Å resolution. The structure showed some remarkably distinctive aspects compared with other closely related Immunoglobulin domains. The structure illustrated the relative orientation of two domains and highlighted the role of the linker region in the domain orientation. We also observed an apparent electron density of Ca2+ ions coordinated with a proper interacting geometry within the protein. Molecular dynamic simulations demonstrated the involvement of a linker salt bridge in providing rigidity between the two domains. Our study proposes an overall arrangement of Ig-like domains in the LigA protein. The structural understanding of the extracellular portion of LigA and its interaction with the ECM provides insight into developing new therapeutics directed toward leptospirosis.

Use of an Integrated Multi-Omics Approach To Identify Molecular Mechanisms and Critical Factors Involved in the Pathogenesis of Leptospira.

Leptospirosis, a bacterial zoonosis caused by pathogenic Leptospira spp., is prevalent worldwide and has become a serious threat in recent years. Limited understanding of Leptospira pathogenesis and host response has hampered the development of effective vaccine and diagnostics. Although Leptospira is phagocytosed by innate immune cells, it resists its destruction, and the evading mechanism involved is unclear. In the present study, we used an integrative multi-omics approach to identify the critical molecular factors of Leptospira involved in pathogenesis during interaction with human macrophages. Transcriptomic and proteomic analyses were performed at 24 h postinfection of human macrophages (phorbol-12-myristate-13-acetate differentiated THP-1 cells) with the pathogenic Leptospira interrogans serovar Icterohaemorrhagiae strain RGA (LEPIRGA). Our results identified a total of 1,528 transcripts and 871 proteins that were significantly expressed with an adjusted P value of <0.05. The correlations between the transcriptomic and proteomic data were above average (r = 0.844), suggesting the role of the posttranscriptional processes during host interaction. The conjoint analysis revealed the expression of several virulence-associated proteins such as adhesins, invasins, and secretory and chemotaxis proteins that might be involved in various processes of attachment and invasion and as effectors during pathogenesis in the host. Further, the interaction of bacteria with the host cell (macrophages) was a major factor in the differential expression of these proteins. Finally, eight common differentially expressed RNA-protein pairs, predicted as virulent, outer membrane/extracellular proteins were validated by quantitative PCR. This is the first report using integrated multi-omics approach to identify critical factors involved in Leptospira pathogenesis. Validation of these critical factors may lead to the identification of target antigens for the development of improved diagnostics and vaccines against leptospirosis. IMPORTANCE Leptospirosis is a zoonotic disease of global importance. It is caused by a Gram-negative bacterial spirochete of the genus Leptospira. The current challenge is to detect the infection at early stage for treatment or to develop potent vaccines that can induce cross-protection against various pathogenic serovars. Understanding host-pathogen interactions is important to identify the critical factors involved in pathogenesis and host defense for developing improved vaccines and diagnostics. Utilizing an integrated multi-omics approach, our study provides important insight into the interaction of Leptospira with human macrophages and identifies a few critical factors (such as virulence-associated proteins) involved in pathogenesis. These factors can be exploited for the development of novel tools for the detection, treatment, or prevention of leptospirosis.

The structure and functional mechanism of eyespot in Chlamydomonas.

Light plays a crucial role in photosynthesis, photoperiodism, and photomorphogenesis. Algae have a specialized visual system to perceive the light signal known as eyespot. A typical eyespot is an orange-colored, membranous structure packed with pigmented granules. In algae, the eyespot membrane bears a specialized type of photoreceptors, which shows similarity with animal rhodopsin photoreceptors. This light-sensing receptor is responsible for the photo-mobility response known as phototaxis. In this, light acts as a signal for onset and cascade of downstream signal transduction pathway leading to a conformational change in photoreceptor. This induces the continuous influx of calcium ions through the opening of calcium ion channels leading to membrane depolarization, and beating of flagella which is responsible for phototaxis. Mutational studies have assisted the discovery of eyespot genes, which are involved in eyespot development, assembly, size control, and functioning in Chlamydomonas. These genes belong to photoreceptors (cop1-12, acry, pcry, cry-dash1, cry-dash2, phot, uvr8), eyeless mutants (eye2, eye3), miniature-eyespot mutants (min1, min2), multiple eyespot mutants (mlt1, mlt2). This review discusses the structural biology of eyespots with special reference to Chlamydomonas, molecular insights, related genes, and proteins responsible for its proper functioning.

Anti-CRISPR proteins as a therapeutic agent against drug-resistant bacteria.

The continuous deployment of various antibiotics to treat multiple serious bacterial infections leads to multidrug resistance among the bacterial population. It has failed the standard treatment strategies through different antibacterial agents and serves as a significant threat to public health worldwide at devastating levels. The discovery of anti-CRISPR proteins catches the interest of researchers around the world as a promising therapeutic agent against drug-resistant bacteria. Anti-CRISPR proteins are known to inhibit bacterial CRISPR-Cas defense systems in multiple possible ways. The CRISPR-Cas nucleoprotein assembly provides adaptive immunity in bacteria against diverse categories of phage infections. Parallelly, phages also try to break the CRISPR-Cas barrier by producing anti-CRISPR proteins, leading to growth inhibition and bacterial lysis. This review begins with a brief description of the bacterial CRISPR-Cas system, followed by a detailed portrayal of anti-CRISPR proteins, including their discovery and evolution, mechanism of action, regulation of expression, and potential applications in the healthcare sector as an alternative therapeutic strategy to combat severe bacterial infections.

Deciphering the Role of Leptospira Surface Protein LigA in Modulating the Host Innate Immune Response.

Leptospira, a zoonotic pathogen, is known to infect various hosts and can establish persistent infection. This remarkable ability of bacteria is attributed to its potential to modulate (activate or evade) the host immune response by exploiting its surface proteins. We have identified and characterized the domain of the variable region of Leptospira immunoglobulin-like protein A (LAV) involved in immune modulation. The 11th domain (A11) of the variable region of LigA (LAV) induces a strong TLR4 dependent innate response leading to subsequent induction of humoral and cellular immune responses in mice. A11 is also involved in acquiring complement regulator FH and binds to host protease Plasminogen (PLG), there by mediating functional activity to escape from complement-mediated killing. The deletion of A11 domain significantly impaired TLR4 signaling and subsequent reduction in the innate and adaptive immune response. It also inhibited the binding of FH and PLG thereby mediating killing of bacteria. Our study discovered an unprecedented role of LAV as a nuclease capable of degrading Neutrophil Extracellular Traps (NETs). This nuclease activity was primarily mediated by A11. These results highlighted the moonlighting function of LigA and demonstrated that a single domain of a surface protein is involved in modulating the host innate immune defenses, which might allow the persistence of Leptospira in different hosts for a long term without clearance.