Specificity and sensitivity of an RNA targeting type III CRISPR complex coupled with a NucC endonuclease effector

Type III CRISPR systems detect invading RNA, resulting in the activation of the enzymatic Cas10 subunit. The Cas10 cyclase domain generates cyclic oligoadenylate (cOA) second messenger molecules, activating a variety of effector nucleases that degrade nucleic acids to provide immunity. The prophage-encoded Vibrio metoecus type III-B (VmeCmr) locus is uncharacterised, lacks the HD nuclease domain in Cas10 and encodes a NucC DNA nuclease effector that is also found associated with Cyclic-oligonucleotide-based anti-phage signalling systems (CBASS). Here we demonstrate that VmeCmr is activated by target RNA binding, generating cyclic-triadenylate (cA₃) to stimulate a robust NucC-mediated DNase activity. The specificity of VmeCmr is probed, revealing the importance of specific nucleotide positions in segment 1 of the RNA duplex and the protospacer flanking sequence (PFS). We harness this programmable system to demonstrate the potential for a highly specific and sensitive assay for detection of the SARS-CoV-2 virus RNA with a limit of detection (LoD) of 2 fM using a commercial plate reader without any extrinsic amplification step. The sensitivity is highly dependent on the guide RNA used, suggesting that target RNA secondary structure plays an important role that may also be relevant in vivo.     

InvB is a type III secretion-associated chaperone for the Salmonella enterica effector protein SopE

SopE is a bacteriophage-encoded effector protein of Salmonella enterica serovar Typhimurium that is translocated into the cytosol of eukaryotic cells by a type III secretion system (TTSS) (W.-D. Hardt, H. Urlaub, and J. E. Galán, Proc. Natl. Acad. Sci. USA 95:2574-2579, 1998; M. W. Wood, R. Rosqvist, P. B. Mullan, M. H. Edwards, and E. E. Galyov, Mol. Microbiol. 22:327-338, 1996). In this study, we provide evidence that an unlinked gene carried within the Salmonella pathogenicity island 1 (SPI-1), invB (K. Eichelberg, C. Ginocchio, and J. E. Galán, J. Bacteriol. 176:4501-4510, 1994), is required for the secretion of SopE through the SPI-1 TTSS. Furthermore, far-Western blotting analysis shows that SopE directly interacts with InvB through a domain located at its amino terminus. We conclude that InvB is the TTSS-associated chaperone for SopE.     

Genetic and molecular evidence that the Pseudomonas syringae type III effector protein AvrRpt2 is a cysteine protease

Upon delivery to the plant cell during infection, the Pseudomonas syringae effector protein AvrRpt2 undergoes proteolytic processing, enhances pathogen virulence and causes the elimination of the Arabidopsis RIN4 protein. A structure-prediction method was employed in order to investigate possible biochemical functions of AvrRpt2. Results of a secondary structure prediction algorithm suggest that the functional C-terminal portion of AvrRpt2 is a cysteine protease. Mutation of predicted catalytic residues within this portion of AvrRpt2 abolished in planta processing, elimination of Arabidopsis RIN4, and the ability to trigger an RPS2-specific resistance response. These data indicate that AvrRpt2 is most likely a sequence divergent cysteine protease whose activity is required for elimination of RIN4 during infection.     

Shigella enterotoxin-2 is a type III effector that participates in Shigella-induced interleukin 8 secretion by epithelial cells

We have previously described a protein termed Shigella enterotoxin 2 (ShET-2), which induces rises in short-circuit current in rabbit ileum mounted in the Ussing chamber. Published reports have postulated that ShET-2 may be secreted by the Shigella type III secretion system (T3SS). In this study, we show that ShET-2 secretion into the extracellular space requires the T3SS in Shigella flexneri 2a strain 2457T and a ShET-2-TEM fusion was translocated into epithelial cells in a T3SS-dependent manner. The ShET-2 gene, sen, is encoded downstream of the ospC1 gene of S. flexneri, and we show that sen is cotranscribed with this T3SS-secreted product. Considering that T3SS effectors have diverse roles in Shigella infection and that vaccine constructs lacking ShET-2 are attenuated in volunteers, we asked whether ShET-2 has a function other than its enterotoxic activity. We constructed a ShET-2 mutant in 2457T and tested its effect on epithelial cell invasion, plaque formation, guinea pig keratoconjunctivitis and interleukin 8 (IL-8) secretion from infected monolayers. Although other phenotypes were not different compared with the wild-type parent, we found that HEp-2 and T84 cells infected with the ShET-2 mutant exhibited significantly reduced IL-8 secretion into the basolateral compartment, suggesting that ShET-2 might participate in the Shigella-induced inflammation of epithelial cells.     

The EHEC type III effector NleL is an E3 ubiquitin ligase that modulates pedestal formation

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes hemorrhagic colitis and may result in potentially fatal hemolytic uremia syndrome in humans. EHEC colonize the intestinal mucosa and promote the formation of actin-rich pedestals via translocated type III effectors. Two EHEC type III secreted effectors, Tir and EspFu/TccP, are key players for pedestal formation. We discovered that an EHEC effector protein called Non-LEE-encoded Ligase (NleL) is an E3 ubiquitin ligase. In vitro, we showed that the NleL C753 residue is critical for its E3 ligase activity. Functionally, we demonstrated that NleL E3 ubiquitin ligase activity is involved in modulating Tir-mediated pedestal formation. Surprisingly, EHEC mutant strain deficient in the E3 ligase activity induced more pedestals than the wild-type strain. The canonical EPEC strain E2348/69 normally lacks the nleL gene, and the ectopic expression of the wild-type EHEC nleL, but not the catalytically-deficient nleL(C753A) mutant, in this strain resulted in fewer actin-rich pedestals. Furthermore, we showed that the C. rodentium NleL homolog is a E3 ubiquitin ligase and is required for efficient infection of murine colonic epithelial cells in vivo. In summary, our study demonstrated that EHEC utilizes NleL E3 ubiquitin ligase activity to modulate Tir-mediated pedestal formation.     

The discovery of SycO highlights a new function for type III secretion effector chaperones

Bacterial injectisomes deliver effector proteins straight into the cytosol of eukaryotic cells (type III secretion, T3S). Many effectors are associated with a specific chaperone that remains inside the bacterium when the effector is delivered. The structure of such chaperones and the way they interact with their substrate is well characterized but their main function remains elusive. Here, we describe and characterize SycO, a new chaperone for the Yersinia effector kinase YopO. The chaperone-binding domain (CBD) within YopO coincides with the membrane localization domain (MLD) targeting YopO to the host cell membrane. The CBD/MLD causes intrabacterial YopO insolubility and the binding of SycO prevents this insolubility but not folding and activity of the kinase. Similarly, SycE masks the MLD of YopE and SycT covers an aggregation-prone domain of YopT, presumably corresponding to its MLD. Thus, SycO, SycE and most likely SycT mask, inside the bacterium, a domain needed for proper localization of their cognate effector in the host cell. We propose that covering an MLD might be an essential function of T3S effector chaperones.     

BopC is a novel type III effector secreted by Bordetella bronchiseptica and has a critical role in type III-dependent necrotic cell death

In Bordetella bronchiseptica, the functional type III secretion system (TTSS) is required for the induction of necrotic cell death in infected mammalian cells. To identify the factor(s) involved in necrotic cell death, type III-secreted proteins from B. bronchiseptica were analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and electrospray ionization tandem mass spectrometry. We identified a 69-kDa secreted protein designated BopC. The gene encoding BopC is located outside of the TTSS locus and is also highly conserved in both Bordetella parapertussis and Bordetella pertussis. The results of a lactate dehydrogenase release assay and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling assay demonstrated that BopC is required for necrotic cell death. It has been reported that tyrosine-phosphorylated proteins (PY) of host cells are dephosphorylated during B. bronchiseptica infection in a TTSS-dependent manner. We found that BopC is also involved in PY dephosphorylation in infected host cells. It appears that the necrotic cell death triggered by BopC occurs prior to the PY reduction in host cells, because Bordetella-induced cell death was not affected even in the presence of a dephosphorylation inhibitor. Furthermore, a translocation assay showed that the signal sequence for both secretion into culture supernatant and translocation into the host cell is located in 48 amino acid residues of the BopC N terminus. This report reveals for the first time that a novel type III effector, BopC, is required for the induction of necrotic cell death during Bordetella infection.     

The Shigella type III effector IpgD recodes Ca2+ signals during invasion of epithelial cells

The role of second messengers in the diversion of cellular processes by pathogens remains poorly studied despite their importance. Among these, Ca²⁺ virtually regulates all known cell processes, including cytoskeletal reorganization, inflammation, or cell death pathways. Under physiological conditions, cytosolic Ca²⁺ increases are transient and oscillatory, defining the so‐called Ca²⁺ code that links cell responses to specific Ca²⁺ oscillatory patterns. During cell invasion, Shigella induces atypical local and global Ca²⁺ signals. Here, we show that by hydrolyzing phosphatidylinositol‐(4,5)bisphosphate, the Shigella type III effector IpgD dampens inositol‐(1,4,5)trisphosphate (InsP₃) levels. By modifying InsP₃ dynamics and diffusion, IpgD favors the elicitation of long‐lasting local Ca²⁺ signals at Shigella invasion sites and converts Shigella‐induced global oscillatory responses into erratic responses with atypical dynamics and amplitude. Furthermore, IpgD eventually inhibits InsP₃‐dependent responses during prolonged infection kinetics. IpgD thus acts as a pathogen regulator of the Ca²⁺ code implicated in a versatility of cell functions. Consistent with this function, IpgD prevents the Ca²⁺‐dependent activation of calpain, thereby preserving the integrity of cell adhesion structures during the early stages of infection.     

SopD2 is a novel type III secreted effector of Salmonella typhimurium that targets late endocytic compartments upon delivery into host cells

Salmonella typhimurium is a facultative intracellular pathogen that utilizes two type III secretion systems to deliver virulence proteins into host cells. These proteins, termed effectors, alter host cell function to allow invasion into and intracellular survival/replication within a vacuolar compartment. Here we describe SopD2, a novel member of the Salmonella translocated effector (STE) family, which share a conserved N-terminal type III secretion signal. Disruption of the sopD2 gene prolonged the survival of mice infected with a lethal dose of Salmonella typhimurium , demonstrating a significant role for this effector in pathogenesis. Expression of sopD2 was induced inside host cells and was dependent on functional ssrA/B and phoP/Q, two component regulatory systems. HA-tagged SopD2 was delivered into HeLa cells in a SPI-2-dependent manner and associated with both the Salmonella -containing vacuole and with swollen endosomes elsewhere in the cell. Subcellular fractionation confirmed that SopD2 was membrane associated in host cells, while the closely related effector SopD was localized to the cytosol. A SopD2 fusion to GFP associated with small tubular structures and large vesicles containing late endocytic markers, including Rab7. Surprisingly, expression of N-terminal amino acids 1–150 of SopD2 fused to GFP was sufficient to mediate both binding to late endosomes/lysosomes and swelling of these compartments. These findings demonstrate that the N-terminus of SopD2 is a bifunctional domain required for both type III secretion out of Salmonella as well as late endosome/lysosome targeting following translocation into host cells .     

Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas immune system

Clustered regularly interspaced short palindromic repeat (CRISPR) is a recently discovered adaptive prokaryotic immune system that provides acquired immunity against foreign nucleic acids by utilizing small guide crRNAs (CRISPR RNAs) to interfere with invading viruses and plasmids. In Escherichia coli, Cas3 is essential for crRNA-guided interference with virus proliferation. Cas3 contains N-terminal HD phosphohydrolase and C-terminal Superfamily 2 (SF2) helicase domains. Here, we provide the first report of the cloning, expression, purification and in vitro functional analysis of the Cas3 protein of the Streptococcus thermophilus CRISPR4 (Ecoli subtype) system. Cas3 possesses a single-stranded DNA (ssDNA)-stimulated ATPase activity, which is coupled to unwinding of DNA/DNA and RNA/DNA duplexes. Cas3 also shows ATP-independent nuclease activity located in the HD domain with a preference for ssDNA substrates. To dissect the contribution of individual domains, Cas3 separation-of-function mutants (ATPase(+)/nuclease(-) and ATPase(-)/nuclease(+)) were obtained by site-directed mutagenesis. We propose that the Cas3 ATPase/helicase domain acts as a motor protein, which assists delivery of the nuclease activity to Cascade-crRNA complex targeting foreign DNA.     

VopV, an F-actin-binding type III secretion effector, is required for Vibrio parahaemolyticus-induced enterotoxicity

Vibrio parahaemolyticus, a Gram-negative halophilic bacterium that causes acute gastroenteritis in humans, is characterized by two type III secretion systems (T3SS), namely T3SS1 and T3SS2. T3SS2 is indispensable for enterotoxicity but the effector(s) involved are unknown. Here, we identify VopV as a critical effector that is required to mediate V. parahaemolyticus T3SS2-dependent enterotoxicity. VopV was found to possess multiple F-actin-binding domains and the enterotoxicity caused by VopV correlated with its F-actin-binding activity. Furthermore, a T3SS2-related secretion system and a vopV homologous gene were also involved in the enterotoxicity of a non-O1/non-O139 V. cholerae strain. These results indicate that the F-actin-targeting effector VopV is involved in enterotoxic activity of T3SS2-possessing bacterial pathogens.     

The evolution of Pseudomonas syringae host specificity and type III effector repertoires

The discovery 45 years ago that many Pseudomonas syringae pathovars elicit the hypersensitive response in plant species other than their hosts fostered the use of these bacteria as experimental models. However, the basis for host specificity and the corresponding resistance of nonhosts remain unclear. Pseudomonas syringae is now known to inject into the host cytoplasm, via the type III secretion system, effector proteins that suppress basal innate immunity, but may be recognized by cognate resistance (R) proteins in a second level of defence. The identification and manipulation of complete repertoires of type III effectors have revealed the highly polymorphic nature of effector repertoires and their potential to limit the host range. However, the maintenance of compatible effector repertoires may be driven by adaptations to life in a given plant species involving many factors. Tools are now available to test several hypotheses for the nature and evolution of P. syringae host specificity and nonhost resistance.     

The targeting of plant cellular systems by injected type III effector proteins

The battle between phytopathogenic bacteria and their plant hosts has revealed a diverse suite of strategies and mechanisms employed by the pathogen or the host to gain the higher ground. Pathogens continually evolve tactics to acquire host resources and dampen host defences. Hosts must evolve surveillance and defence systems that are sensitive enough to rapidly respond to a diverse range of pathogens, while reducing costly and damaging inappropriate misexpression. The primary virulence mechanism employed by many bacteria is the type III secretion system, which secretes and translocates effector proteins directly into the cells of their plant hosts. Effectors have diverse enzymatic functions and can target specific components of plant systems. While these effectors should favour bacterial fitness, the host may be able to thwart infection by recognizing the activity or presence of these foreign molecules and initiating retaliatory immune measures. We review the diverse host cellular systems exploited by bacterial effectors, with particular focus on plant proteins directly targeted by effectors. Effector–host interactions reveal different stages of the battle between pathogen and host, as well as the diverse molecular strategies employed by bacterial pathogens to hijack eukaryotic cellular systems.     

Function and distribution of EspG2, a type III secretion system effector of enteropathogenic Escherichia coli

The enteropathogenic Escherichia coli (EPEC) effector protein EspG, like the Shigella effector VirA, functions through disruption of the host cell microtubule network. Reports have differed as to whether the EspG homologue, EspG2, is also responsible for microtubule disruption. In this study we show that following translocation, EspG2 and VirA are localised under adherent bacteria and able to restore the microtubule disruption phenotype to an espG/espG2 double EPEC mutant. The espG/espG2 double mutant produced A/E lesions similar to wild-type EPEC on human intestinal in vitro organ cultures. Determining the distribution of espG and espG2 among clinical EPEC isolates revealed two different types of espG (espG alpha and espG beta) and espG2 (intact and pseudo genes), which were associated with specific EPEC serotypes and closely followed the EPEC lineage. This investigation has established a role for EspG2 in the disruption of the microtubule network and associated different espG and espG2 types with different groups of EPEC.     

Identification of a bacterial type III effector family with G protein mimicry functions

Many bacterial pathogens use the type III secretion system to inject "effector" proteins into host cells. Here, we report the identification of a 24 member effector protein family found in pathogens including Salmonella, Shigella, and enteropathogenic E. coli. Members of this family subvert host cell function by mimicking the signaling properties of Ras-like GTPases. The effector IpgB2 stimulates cellular responses analogous to GTP-active RhoA, whereas IpgB1 and Map function as the active forms of Rac1 and Cdc42, respectively. These effectors do not bind guanine nucleotides or have sequences corresponding the conserved GTPase domain, suggesting that they are functional but not structural mimics. However, several of these effectors harbor intracellular targeting sequences that contribute to their signaling specificities. The activities of IpgB2, IpgB1, and Map are dependent on an invariant WxxxE motif found in numerous effectors leading to the speculation that they all function by a similar molecular mechanism.     

The Pseudomonas syringae type III effector HopAM1 enhances virulence on water-stressed plants

Pseudomonas syringae strains deliver diverse type III effector proteins into host cells, where they can act as virulence factors. Although the functions of the majority of type III effectors are unknown, several have been shown to interfere with plant basal defense mechanisms. Type III effectors also could contribute to bacterial virulence by enhancing nutrient uptake and pathogen adaptation to the environment of the host plant. We demonstrate that the type III effector HopAM1 (formerly known as AvrPpiB) enhances the virulence of a weak pathogen in plants that are grown under drought stress. This is the first report of a type III effector that aids pathogen adaptation to water availability in the host plant. Expression of HopAM1 makes transgenic Ws-0 Arabidopsis hypersensitive to abscisic acid (ABA) for stomatal closure and germination arrest. Conditional expression of HopAM1 in Arabidopsis also suppresses basal defenses. ABA responses overlap with defense responses and ABA has been shown to suppress defense against P. syringae pathogens. We propose that HopAM1 aids P. syringae virulence by manipulation of ABA responses that suppress defense responses. In addition, host ABA responses enhanced by type III delivery of HopAM1 protect developing bacterial colonies inside leaves from osmotic stress.     

Esterification of cholesterol by a type III secretion effector during intracellular Salmonella infection

Survival of Salmonella typhimurium within a vacuole in host cells depends on secreted virulence factors encoded by the Salmonella pathogenicity island 2 (SPI-2). High levels of cholesterol are detected at the Salmonella -containing vacuole (SCV). Here we show that the SPI-2 effector SseJ esterifies cholesterol in vitro , in cells and during infection. Intracellular infections with wild-type as compared with Δ sseJ bacteria led to higher levels of cholesterol ester production in HeLa cells and RAW macrophages and were shown to increase levels of lipid droplets (structures enriched in cholesterol esters). Ectopic expression of SseJ reduced cholesterol levels in cellular membranes and antagonized a major membrane activity of a second bacterial effector known to be important to the stability of the SCV. Previous studies in mouse models of infection have established a virulence defect in Δ sseJ bacteria and have suggested a role for SseJ in regulating SCV dynamics. Our data indicating the molecular activity of SseJ suggest that cholesterol and its esterification at the SCV are functionally important for intracellular bacterial survival.