The Orth Lab is interested in elucidating the activity of virulence factors from pathogenic bacteria. We aim to gain novel molecular insight into the virulence mechanisms caused by effectors as well as the eukaryotic signaling systems they emulate or target.
Many virulence factors are secreted by bacteria using a type III secretion system (T3SS). The T3SS resembles a needle-like structure that efficiently ejects effector proteins from bacteria into the cytosol of a host cell. Effectors have evolved in a manner similar to many viral oncogenes; a eukaryotic activity is usurped and modified by the pathogen for its own advantage.
We are working on bacterial T3SS systems, their regulators and their secreted effectors to understand how signaling systems in the eukaryotic host can be manipulated by bacterial pathogens. These studies provide novel insight into the molecular workings of eukaryotic signal transduction. Our work at UT Southwestern is accomplished using a broad range of tools, including biochemistry, molecular microbiology, protein chemistry, structural biology, yeast genetics, cell biology, and more.
We study the virulence mechanisms of Vibrio parahaemolyticus (pictured above using confocal microscopy, infecting HeLa cells). V. parahaemolyticus is a Gram-negative bacterium commonly found in marine and estuarine environments. Infection typically results from consumption of contaminated shellfish and leads to acute gastroenteritis. Individuals who are immunocompromised or burdened with pre-existing health conditions are at high risk for severe, potentially lethal complications. The V. parahaemolyticus genome contains two pathogenicity island - encoded type III secretion systems (T3SS1 and T3SS2), with corresponding effectors (Vops) that contribute to its pathogenicity.
Vibrio T3SS1
Our studies demonstrate V. parahaemolyticus uses T3SS1 to orchestrate host cell death by autophagy induction, cell blebbing, cell rounding, and then cell lysis. We found deleting one effector, VopQ, completely abolished autophagy induction in infected cells (Burdette et al., Mol Microbiol 2009). The next step entails collapse of the actin cytoskeleton with synchronized rounding of infected cells. We attributed the cell rounding to VopS, an effector that AMPylates the Rho family of GTPases, thereby preventing them from interacting with downstream signaling molecules (Yarbrough et al., Science 2009). Another T3SS1 effector VPA0450 induces membrane blebbing and facilitate host cell lysis via its inositol polyphosphate 5-phosphatase activity that disrupts the dynamic interaction of the cell membrane with the actin cytoskeleton (Broberg et al., Science 2010). Many other effectors are thought to contribute to V. parahaemolyticus - mediated cell death. Future investigations will be directed at understanding the molecular mechanisms they employ to disrupt host cell signaling and promote infection.
Vibrio T3SS1 orchestrates host cell death
Vibrio T3SS2 establishes a protective intracellular niche
Vibrio T3SS2
The Vibrio parahaemolyticus genome contains a second pathogenicity island - encoded type III secretion system (T3SS2) that is associated with enterotoxicity. Since its discovery, this bacterium has been regarded as being exclusive extracellular. However, we demonstrated that V. parahaemolyticus is actually a facultative intracellular pathogen. The novel understanding of V. parahaemolyticus as an intracellular bacterium compares to a “rediscovery” that presents itself as a model poised for future studies of T3SS-mediated disruption of intracellular processes. Putative effectors have since been reassessed in this light and have been shown to contribute to the bacterium’s intracellular lifestyle. We have shown VopC deamidation activity on target host cell GTPases is required for this intracellular invasion (Zhang et al,, Cell Rep. 2012). However, this activity can not be exchanged with a homologous effector from E. coli (CNF1) that also deamidates small GTPases without compromising the invasion process (Lafrance et al., Mbio. 2022).
Ampylation
We discovered a bacterial virulence factor from Vibrio parahaemolyticus that modifies a conserved threonine residue on eukaryotic substrates via a phosphodiester bond with AMP (Yarbrough et al., Science 2009). This modification, called AMPylation, is mediated by a domain called filamentation induced by cAMP (Fic). We have solved the structure of the VopS virulence factor Fic domain and characterized the kinetics of AMPylation as a direct transfer mechanism. As Fic domains are evolutionarily conserved in metazoans, we demonstrated this post-translational modification in eukaryotes. Fic is localized in the ER and reversably AMPylates BiP/GRP78 to maintain ER homeostasis. Further studies demonstrated that adaptation to constant light requires Fic-mediated AMPylation of BiP to protect against reversible photoreceptor degeneration.
Fic-mediated AMPylation of BiP tempers the unfolded protein response
Other Research
The rice blast fungus, Magnaporthe oryzae, causes one of the most destructive diseases of cultivated rice in the world. Infections caused by this recalcitrant pathogen leads to the annual destruction of approximately 10-30% of the rice harvested globally. We are interested in understanding virulence factors that contribute to host pathogenesis.
Cancer evolves through a multistep process that occurs by the temporal accumulation of genetic mutations. Tumor-derived exosomes are emerging contributors to tumorigenesis. To understand how exosomes might contribute to cell transformation, we utilized the classic two-step NIH/3T3 cell transformation assay and observed that exosomes isolated from pancreatic cancer cells, but not normal human cells, can initiate malignant cell transformation and these transformed cells formed tumors in vivo. However, cancer cell exosomes are unable to transform cells alone or to act as a promoter of cell transformation. Utilizing proteomics and exome sequencing, we discovered cancer cell exosomes act as an initiator by inducing random mutations in recipient cells. Cells from the pool of randomly mutated cells are driven to transformation by a classic promoter resulting in foci, each of which encode a unique genetic profile. Our studies describe a novel molecular understanding of how cancer cell exosomes contribute to cell transformation.
We have identified a factor involved in initial binding of host cells by a wide range of Gram-negative pathogens, Multivalent Adhesion Molecule (MAM) 7. Interference with MAM7-mediated attachment, by pre-incubation of host cells with recombinant MAM7, significantly delays the onset of hallmarks of infection. Targeting or exploiting MAM7 might provide a valuable tool for combating Gram-negative bacterial infections and we are currently exploring applications of MAM7 as an antimicrobial agent.