Structural investigation on SPI-6-associated Salmonella typhimurium VirG-like stress protein that promotes pathogen survival in macrophages.

Enteric microbial pathogenesis, remarkably a complex process, is achieved by virulence factors encoded by genes located within regions of the bacterial genome termed pathogenicity islands. Salmonella pathogenicity islands (SPI) encodes proteins, that are essential virulence determinants for pathogen colonization and virulence. In addition to the well-characterized SPI-1 and SPI-2 proteins, which are required for bacterial invasion and intracellular replication, respectively, SPI-6 (formerly known as Salmonella enterica centisome 7 island [SCI]) encoding proteins are also known to play pivotal role in Salmonella pathogenesis. However, the underlying molecular mechanism of these proteins remained elusive. To gain molecular insights into SPI-6-associated proteins, in this study, a SPI-6 Salmonella typhimurium VirG-like protein (STV) is characterized using interdisciplinary experimental approaches including X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy and infection assays. The high-resolution crystal structure, determined by the single-wavelength anomalous dispersion (SAD) method, reveals that STV belongs to the LTxxQ motif family. Solution-state NMR spectroscopy studies reveal that STV form a dimer involving interconnected helices. Interestingly, functional studies show that STV influence pathogen persistence inside macrophages in vitro at later stages of infection. Altogether, our findings suggest that STV, a member of the LTxxQ stress protein family, modulates bacterial survival mechanism in macrophages through SPI-1 and SPI-2 genes, respectively.

Crystal structure of the usher chaperone YadV reveals a monomer with the proline lock in closed conformation suggestive of an intermediate state.

Cell surface pili assembled by the chaperone-usher (CU) pathway play a crucial role in the adhesion of uropathogenic Escherichia coli. YadV is the chaperone component of the CU pathway of Yad pili. Here, we report the crystal structure of YadV from E. coli. In contrast to major usher chaperones, YadV is a monomer in solution as well as in the crystallographic symmetry, and the monomeric form is a preferred state for interacting with pilus subunits. Moreover, we observed a closed conformation for the proline lock, a crucial structural element for chaperone-pilus subunit interaction. MD simulation shows that the closed state of the proline lock is not energetically stable. Thus, the structure of monomeric YadV with its closed proline lock may serve as an intermediate state to provide suitable access to pilus subunits.

Cloning, purification, crystallization and preliminary X-ray diffraction studies of Escherichia coli PapD-like protein (EcpD).

Many Gram-negative bacteria are characterized by hair-like proteinaceous appendages on their surface known as fimbriae. In uropathogenic strains of Escherichia coli, fimbriae mediate attachment by binding to receptors on the host cell, often contributing to virulence and disease. E. coli PapD-like protein (EcpD) is a periplasmic chaperone that plays an important role in the proper folding and guiding of Yad fimbrial proteins to the outer membrane usher protein in a process known as pilus biogenesis. EcpD is essential for pilus biogenesis in uropathogenic E. coli and plays an important role in virulence. In the present study, EcpD was cloned, overexpressed, purified and crystallized by the hanging-drop vapour-diffusion method. The crystals diffracted to 1.67 Šresolution and belonged to the orthorhombic space group C222(1), with unit-cell parameters a = 100.3, b = 127.6, c = 45.9 Å. There was a single molecule in the asymmetric unit and the corresponding Matthews coefficient was calculated to be 3.02 Å(3) Da(-1), with 59% solvent content. Initial phases were determined by molecular replacement.

CD4+ T cell-derived novel peptide Thp5 induces interleukin-4 production in CD4+ T cells to direct T helper 2 cell differentiation.

The differentiation of naïve CD4(+) T cells into T helper 2 (Th2) cells requires production of the cytokine IL-4 in the local microenvironment. It is evident that naïve/quiescently activated CD4(+) T cells produce the IL-4 that drives Th2 cell differentiation. Because early production of IL-4 in naïve T cells leads to preferential Th2 cell differentiation, this process needs to be tightly regulated so as to avoid catastrophic and misdirected Th2 cell differentiation. Here, we show that Thp5, a novel peptide with structural similarity to vasoactive intestinal peptide, regulates production of early IL-4 in newly activated CD4(+) T cells. Induction of IL-4 in CD4(+) T cells by Thp5 is independent of the transcription factor STAT6 but dependent on ERK1/2 signaling. Furthermore, cytokines (IL-12 and TGF-ß) that promote the differentiation of Th1 or Th17 cells inhibit Thp5 induction, thus suppressing Th2 cell differentiation. We further showed that Thp5 enhances Th2 responses and exacerbates allergic airway inflammation in mice. Taken together, our findings reveal that early activated CD4(+) T cells produce Thp5, which plays a critical role as a molecular switch in the differentiation of Th cells, biasing the response toward the Th2 cell phenotype.