Composition and Biophysical Properties of the Sorting Platform Pods in the Type III Secretion System.

, causative agent of bacillary dysentery (shigellosis), uses a type III secretion system (T3SS) as its primary virulence factor. The T3SS injectisome delivers effector proteins into host cells to promote entry and create an important intracellular niche. The injectisome's cytoplasmic sorting platform (SP) is a critical assembly that contributes to substrate selection and energizing secretion.

The cytoplasmic domain of MxiG interacts with MxiK and directs assembly of the sorting platform in the type III secretion system.

Many Gram-negative bacteria use type III secretion systems (T3SSs) to inject virulence effector proteins into eukaryotic cells. The T3SS apparatus (T3SA) is structurally conserved among diverse bacterial pathogens and consists of a cytoplasmic sorting platform, an envelope-spanning basal body, and an extracellular needle with tip complex. The sorting platform is essential for effector recognition and powering secretion.

The Tip Complex: From Host Cell Sensing to Translocon Formation.

Type III secretion systems are used by some Gram-negative bacteria to inject effector proteins into targeted eukaryotic cells for the benefit of the bacterium. The type III secretion injectisome is a complex nanomachine comprised of four main substructures including a cytoplasmic sorting platform, an envelope-spanning basal body, an extracellular needle and an exposed needle tip complex. Upon contact with a host cell, secretion is induced, resulting in the formation of a translocon pore in the host membrane.

The Loss of Expression of a Single Type 3 Effector (CT622) Strongly Reduces Infectivity and Growth.

Invasion of epithelial cells by the obligate intracellular bacterium results in its enclosure inside a membrane-bound compartment termed an inclusion. The bacterium quickly begins manipulating interactions between host intracellular trafficking and the inclusion interface, diverging from the endocytic pathway and escaping lysosomal fusion. We have identified a previously uncharacterized protein, CT622, unique to the , in the absence of which most bacteria failed to establish a successful infection.

Using disruptive insertional mutagenesis to identify the in situ structure-function landscape of the Shigella translocator protein IpaB.

Bacterial type III secretion systems (T3SS) are used to inject proteins into mammalian cells to subvert cellular functions. The Shigella T3SS apparatus (T3SA) is comprised of a basal body, cytoplasmic sorting platform and exposed needle with needle "tip complex" (TC). TC maturation occurs when the translocator protein IpaB is recruited to the needle tip where both IpaD and IpaB control secretion induction.

Single-domain antibodies pinpoint potential targets within invasion plasmid antigen D of the needle tip complex for inhibition of type III secretion.

Numerous Gram-negative pathogens infect eukaryotes and use the type III secretion system (T3SS) to deliver effector proteins into host cells. One important T3SS feature is an extracellular needle with an associated tip complex responsible for assembly of a pore-forming translocon in the host cell membrane. spp.

Evaluation of lumazine synthase from Bacillus anthracis as a presentation platform for polyvalent antigen display.

Polyvalent antigen display is an effective strategy to enhance the immunogenicity of subunit vaccines by clustering them in an array-like manner on a scaffold system. This strategy results in a higher local density of antigens, increased high avidity interactions with B cells and other antigen presenting cells, and therefore a more effective presentation of vaccine antigens. In this study, we used lumazine synthase (LS), an icosahedral symmetry capsid derived from Bacillus anthracis, as a scaffold to present 60 copies of a linear B cell epitope (PB10) from the ricin toxin fused to the C terminus of LS via four different linkers.

Recombinant Expression and Purification of the Shigella Translocator IpaB.

Type III secretion systems (T3SS) are highly conserved virulence factors employed by a large number of pathogenic gram-negative bacteria. Like many T3SS translocators, recombinant expression of the hydrophobic Shigella protein IpaB requires the presence of its cognate chaperone IpgC. Chaperone-bound IpaB is maintained in a nonfunctional state, which has hampered in vitro studies aimed at understanding molecular structure and function of this important class of T3SS proteins.

Characterization of Type Three Secretion System Translocator Interactions with Phospholipid Membranes.

In vitro characterization of type III secretion system (T3SS) translocator proteins has proven challenging due to complex purification schemes and their hydrophobic nature that often requires detergents to provide protein solubility and stability. Here, we provide experimental details for several techniques that overcome these hurdles, allowing for the direct characterization of the Shigella translocator protein IpaB with respect to phospholipid membrane interaction. The techniques specifically discussed in this chapter include membrane interaction/liposome flotation, liposome sensitive fluorescence quenching, and protein-mediated liposome disruption assays.

Interrogating Genes That Mediate Chlamydia trachomatis Survival in Cell Culture Using Conditional Mutants and Recombination.

Unlabelled: Intracellular bacterial pathogens in the family Chlamydiaceae are causes of human blindness, sexually transmitted disease, and pneumonia. Genetic dissection of the mechanisms of chlamydial pathogenicity has been hindered by multiple limitations, including the inability to inactivate genes that would prevent the production of elementary bodies. Many genes are also Chlamydia-specific genes, and chlamydial genomes have undergone extensive reductive evolution, so functions often cannot be inferred from homologs in other organisms.

Computational modeling of TC0583 as a putative component of the Chlamydia muridarum V-type ATP synthase complex and assessment of its protective capabilities as a vaccine antigen.

Numerous Chlamydia trachomatis proteins have been identified as potential subunit vaccines, of which the major outer-membrane protein (MOMP) has, so far, proven the most efficacious. Recently, subunit A of the V-type ATP synthase (ATPase; TC0582) complex was shown to elicit partial protection against infection. Computational modeling of a neighboring gene revealed a novel subunit of the V-type ATPase (TC0583).

Hypothetical protein CT398 (CdsZ) interacts with σ(54) (RpoN)-holoenzyme and the type III secretion export apparatus in Chlamydia trachomatis.

A significant challenge to bacteriology is the relatively large proportion of proteins that lack sufficient sequence similarity to support functional annotation (i.e. hypothetical proteins).

Chlamydia trachomatis protein CT009 is a structural and functional homolog to the key morphogenesis component RodZ and interacts with division septal plane localized MreB.

Cell division in Chlamydiae is poorly understood as apparent homologs to most conserved bacterial cell division proteins are lacking and presence of elongation (rod shape) associated proteins indicate non-canonical mechanisms may be employed. The rod-shape determining protein MreB has been proposed as playing a unique role in chlamydial cell division. In other organisms, MreB is part of an elongation complex that requires RodZ for proper function.

Structural and biochemical characterization of Chlamydia trachomatis hypothetical protein CT263 supports that menaquinone synthesis occurs through the futalosine pathway.

The obligate intracellular human pathogen Chlamydia trachomatis is the etiological agent of blinding trachoma and sexually transmitted disease. Genomic sequencing of Chlamydia indicated this medically important bacterium was not exclusively dependent on the host cell for energy. In order for the electron transport chain to function, electron shuttling between membrane-embedded complexes requires lipid-soluble quinones (e.

Atypical response regulator ChxR from Chlamydia trachomatis is structurally poised for DNA binding.

ChxR is an atypical two-component signal transduction response regulator (RR) of the OmpR/PhoB subfamily encoded by the obligate intracellular bacterial pathogen Chlamydia trachomatis. Despite structural homology within both receiver and effector domains to prototypical subfamily members, ChxR does not require phosphorylation for dimer formation, DNA binding or transcriptional activation. Thus, we hypothesized that ChxR is in a conformation optimal for DNA binding with limited interdomain interactions.

Chlamydia trachomatis CT771 (nudH) is an asymmetric Ap4A hydrolase.

Asymmetric diadenosine 5',5‴-P(1),P(4)-tetraphosphate (Ap4A) hydrolases are members of the Nudix superfamily that asymmetrically cleave the metabolite Ap4A into ATP and AMP while facilitating homeostasis. The obligate intracellular mammalian pathogen Chlamydia trachomatis possesses a single Nudix family protein, CT771. As pathogens that rely on a host for replication and dissemination typically have one or zero Nudix family proteins, this suggests that CT771 could be critical for chlamydial biology and pathogenesis.

Structure of CT584 from Chlamydia trachomatis refined to 3.05 Å resolution.

Chlamydia trachomatis is a major cause of various diseases, including blinding trachoma and pelvic inflammatory disease, and is the leading reported sexually transmitted bacterial infection worldwide. All pathogenic Chlamydiae spp. utilize a supramolecular syringe, or type III secretion system (T3SS), to inject proteins into their obligate host in order to propagate infection.

Studies of the conformational stability of invasion plasmid antigen B from Shigella.

Shigella spp. are the causative agent of shigellosis, the second leading cause of diarrhea in children of ages 2-5. Despite many years of research, a protective vaccine has been elusive.

Structural basis for nucleotide binding and reaction catalysis in mevalonate diphosphate decarboxylase.

Mevalonate diphosphate decarboxylase (MDD) catalyzes the final step of the mevalonate pathway, the Mg(2+)-ATP dependent decarboxylation of mevalonate 5-diphosphate (MVAPP), producing isopentenyl diphosphate (IPP). Synthesis of IPP, an isoprenoid precursor molecule that is a critical intermediate in peptidoglycan and polyisoprenoid biosynthesis, is essential in Gram-positive bacteria (e.g.

Biochemical and structural basis for inhibition of Enterococcus faecalis hydroxymethylglutaryl-CoA synthase, mvaS, by hymeglusin.

Hymeglusin (1233A, F244, L-659-699) is established as a specific β-lactone inhibitor of eukaryotic hydroxymethylglutaryl-CoA synthase (HMGCS). Inhibition results from formation of a thioester adduct to the active site cysteine. In contrast, the effects of hymeglusin on bacterial HMG-CoA synthase, mvaS, have been minimally characterized.

Identification of the bile salt binding site on IpaD from Shigella flexneri and the influence of ligand binding on IpaD structure.

Type III secretion (TTS) is an essential virulence factor for Shigella flexneri, the causative agent of shigellosis. The Shigella TTS apparatus (TTSA) is an elegant nanomachine that is composed of a basal body, an external needle to deliver effectors into human cells, and a needle tip complex that controls secretion activation. IpaD is at the tip of the nascent TTSA needle where it controls the first step of TTS activation.

The structures of coiled-coil domains from type III secretion system translocators reveal homology to pore-forming toxins.

Many pathogenic Gram-negative bacteria utilize type III secretion systems (T3SSs) to alter the normal functions of target cells. Shigella flexneri uses its T3SS to invade human intestinal cells to cause bacillary dysentery (shigellosis) that is responsible for over one million deaths per year. The Shigella type III secretion apparatus is composed of a basal body spanning both bacterial membranes and an exposed oligomeric needle.

Crystal structures of Staphylococcus epidermidis mevalonate diphosphate decarboxylase bound to inhibitory analogs reveal new insight into substrate binding and catalysis.

The polyisoprenoid compound undecaprenyl phosphate is required for biosynthesis of cell wall peptidoglycans in gram-positive bacteria, including pathogenic Enterococcus, Streptococcus, and Staphylococcus spp. In these organisms, the mevalonate pathway is used to produce the precursor isoprenoid, isopentenyl 5-diphosphate. Mevalonate diphosphate decarboxylase (MDD) catalyzes formation of isopentenyl 5-diphosphate in an ATP-dependent irreversible reaction and is therefore an attractive target for inhibitor development that could lead to new antimicrobial agents.

Evidence for alternative quaternary structure in a bacterial Type III secretion system chaperone.

Background: Type III secretion systems are a common virulence mechanism in many Gram-negative bacterial pathogens. These systems use a nanomachine resembling a molecular needle and syringe to provide an energized conduit for the translocation of effector proteins from the bacterial cytoplasm to the host cell cytoplasm for the benefit of the pathogen. Prior to translocation specialized chaperones maintain proper effector protein conformation.