The polyadenylase PAPI is required for virulence plasmid maintenance in pathogenic bacteria.

Many species of pathogenic bacteria harbor critical plasmid-encoded virulence factors, and yet the regulation of plasmid replication is often poorly understood despite playing a critical role in plasmid-encoded gene expression. Human pathogenic Yersinia, including the plague agent Y. pestis and its close relative Y. pseudotuberculosis, require the type III secretion system (T3SS) virulence factor to subvert host defense mechanisms and colonize host tissues. The Yersinia T3SS is encoded on the IncFII plasmid for Yersinia virulence (pYV). Several layers of gene regulation enables a large increase in expression of Yersinia T3SS genes at mammalian body temperature. Surprisingly, T3SS expression is also controlled at the level of gene dosage. The number of pYV molecules relative to the number of chromosomes per cell, referred to as plasmid copy number, increases with temperature. The ability to increase and maintain elevated pYV plasmid copy number, and therefore T3SS gene dosage, at 37°C is important for Yersinia virulence. In addition, pYV is highly stable in Yersinia at all temperatures, despite being dispensable for growth outside the host. Yet how Yersinia reinforces elevated plasmid replication and plasmid stability remains unclear. In this study, we show that the chromosomal gene pcnB encoding the polyadenylase PAP I is required for regulation of pYV plasmid copy number (PCN), maintenance of pYV in the bacterial population outside the host, robust T3SS activity, and Yersinia virulence in a mouse infection model. Likewise, pcnB/PAP I is also required for robust expression of the Shigella flexneri virulence plasmid-encoded T3SS. Furthermore, Yersinia and Shigella pcnB/PAP I is required for maintaining normal PCN of model antimicrobial resistance (AMR) plasmids whose replication is regulated by sRNA, thereby increasing antibiotic resistance by ten-fold. These data suggest that pcnB/PAP I contributes to the spread and stabilization of virulence and AMR plasmids in bacterial pathogens, and is essential in maintaining the gene dosage required to mediate plasmid-encoded traits. Importantly pcnB/PAP I has been bioinformatically identified in many species of bacteria despite being studied in only a few species to date. Our work highlights the potential importance of pcnB/PAP I in antibiotic resistance, and shows for the first time that pcnB/PAP I reinforces PCN and virulence plasmid stability in natural pathogenic hosts with a direct impact on bacterial virulence.

In situ deposition of nanobodies by an engineered commensal microbe promotes survival in a mouse model of enterohemorrhagic E. coli.

Engineered smart microbes that deliver therapeutic payloads are emerging as treatment modalities, particularly for diseases with links to the gastrointestinal tract. Enterohemorrhagic Escherichia coli (EHEC) is a causative agent of potentially lethal hemolytic uremic syndrome. Given concerns that antibiotic treatment increases EHEC production of Shiga toxin (Stx), which is responsible for systemic disease, novel remedies are needed. EHEC encodes a type III secretion system (T3SS) that injects Tir into enterocytes. Tir inserts into the host cell membrane, exposing an extracellular domain that subsequently binds intimin, one of its outer membrane proteins, triggering the formation of attaching and effacing (A/E) lesions that promote EHEC mucosal colonization. Citrobacter rodentium (Cr), a natural A/E mouse pathogen, similarly requires Tir and intimin for its pathogenesis. Mice infected with Cr(ΦStx2dact), a variant lysogenized with an EHEC-derived phage that produces Stx2dact, develop intestinal A/E lesions and toxin-dependent disease. Stx2a is more closely associated with human disease. By developing an efficient approach to seamlessly modify the C. rodentium genome, we generated Cr_Tir-MEHEC(ΦStx2a), a variant that expresses Stx2a and the EHEC extracellular Tir domain. We found that mouse precolonization with HS-PROT3EcT-TD4, a human commensal E. coli strain (E. coli HS) engineered to efficiently secrete an anti-EHEC Tir nanobody, delayed bacterial colonization and improved survival after challenge with Cr_Tir-MEHEC(ΦStx2a). This study suggests that commensal E. coli engineered to deliver payloads that block essential virulence determinants can be developed as a new means to prevent and potentially treat infections including those due to antibiotic resistant microbes.

In situ deposition of nanobodies by an engineered commensal microbe promotes survival in a mouse model of enterohemorrhagic E. coli.

Engineered smart microbes that deliver therapeutic payloads are emerging as treatment modalities, particularly for diseases with links to the gastrointestinal tract. Enterohemorrhagic E coli (EHEC) is a causative agent of potentially lethal hemolytic uremic syndrome. Given concerns that antibiotic treatment increases EHEC production of Shiga toxin (Stx), which is responsible for systemic disease, novel remedies are needed. EHEC encodes a type III secretion system (T3SS) that injects Tir into enterocytes. Tir inserts into the host cell membrane, exposing an extracellular domain that subsequently binds intimin, one of its outer membrane proteins, triggering the formation of attaching and effacing (A/E) lesions that promote EHEC mucosal colonization. Citrobacter rodentium (Cr), a natural A/E mouse pathogen, similarly requires Tir and intimin for its pathogenesis. Mice infected with Cr(ΦStx2dact), a variant lysogenized with an EHEC-derived phage that produces Stx2dact, develop intestinal A/E lesions and toxin-dependent disease. Stx2a is more closely associated with human disease. By developing an efficient approach to seamlessly modify the C. rodentium genome, we generated Cr_Tir-MEHEC(ΦStx2a), a variant that expresses Stx2a and the EHEC extracellular Tir domain. We found that mouse pre-colonization with HS-PROT3EcT-TD4, a human commensal E. coli strain (E. coli HS) engineered to efficiently secrete- an anti-EHEC Tir nanobody, delayed bacterial colonization and improved survival after challenge with Cr_Tir-MEHEC(ΦStx2a). This study provides the first evidence to support the efficacy of engineered commensal E. coli to intestinally deliver therapeutic payloads that block essential enteric pathogen virulence determinants, a strategy that may serve as an antibiotic-independent antibacterial therapeutic modality.

Gut bacterial type III secretion systems aggravate colitis in mice and serve as biomarkers of Crohn's disease.

BACKGROUND: Mesenteric adipose tissue (mAT) hyperplasia, known as creeping fat, is a pathologic characteristic of Crohn's disease (CD). In our previously reported cohort, we observed that Achromobacter pulmonis was the most abundant and prevalent bacteria cultivated from creeping fat. METHODS: A whole genomic sequencing and identification of T3SS orthologs of mAT-derived A. pulmonis were used. A functional type III secretion system (T3SS) mediated the pathogenic potential of A. pulmonis in vitro and in mouse colitis model. Furthermore, a T3SS Finder pipeline was introduced to evaluate gut bacterial T3SS orthologs in the feces of CD patients, ulcerative colitis and colorectal cancer patients. FINDINGS: Here, we reveal that mAT-derived A. pulmonis possesses a functional T3SS, aggravates colitis in mice via T3SS, and exhibits T3SS-dependent cytotoxicity via a caspase-independent mechanism in macrophages and epithelial cells, which demonstrated the pathogenic potential of the T3SS-harboring A. pulmonis. Metagenomic analyses demonstrate an increased abundance of Achromobacter in the fecal of Crohn's disease patients compared to healthy controls. A comprehensive comparison of total microbial vT3SS abundance in various intestine diseases demonstrated that the specific enrichment of vT3SS genes was shown in fecal samples of CD, neither ulcerative colitis nor colorectal cancer patients, and ten T3SS gene-based biomarkers for CD were discovered and validated in a newly recruited CD cohort. Furthermore, treatment with exclusive enteral nutrition (EEN), an intervention that improves CD patient symptomatology, was found associated with a significant reduction in the prevalence of T3SS genes in fecal samples. INTERPRETATION: These findings highlight the pathogenic significance of T3SSs in the context of CD and identify specific T3SS genes that could potentially function as biomarkers for diagnosing and monitoring the clinical status of CD patients. FUNDING: This work is supported by the National Key Research and Development Program of China (2020YFA0907800), the China Postdoctoral Science Foundation (2023M744089), the National Natural Science Foundation of China (32000096), the Shenzhen Science and Technology Programs (KQTD20200820145822023, RCIC20231211085944057, and ZDSYS20220606100803007), National Key Clinical Discipline, Guangdong Provincial Clinical Research Center for Digestive Diseases (2020B1111170004), Qingfeng Scientific Research Fund of the China Crohn's & Colitis Foundation (CCCF) (CCCF-QF-2022B71-1), and the Sixth Affiliated Hospital, Sun Yat-sen University Clinical Research 1010 Program 1010CG(2023)-08. These funding provided well support for this research work, which involved data collection, analysis, interpretation, patient recruitment and so on.

Engineered Escherichia coli for the in situ secretion of therapeutic nanobodies in the gut.

Drug platforms that enable the directed delivery of therapeutics to sites of diseases to maximize efficacy and limit off-target effects are needed. Here, we report the development of PROT3EcT, a suite of commensal Escherichia coli engineered to secrete proteins directly into their surroundings. These bacteria consist of three modular components: a modified bacterial protein secretion system, the associated regulatable transcriptional activator, and a secreted therapeutic payload. PROT3EcT secrete functional single-domain antibodies, nanobodies (Nbs), and stably colonize and maintain an active secretion system within the intestines of mice. Furthermore, a single prophylactic dose of a variant of PROT3EcT that secretes a tumor necrosis factor-alpha (TNF-α)-neutralizing Nb is sufficient to ablate pro-inflammatory TNF levels and prevent the development of injury and inflammation in a chemically induced model of colitis. This work lays the foundation for developing PROT3EcT as a platform for the treatment of gastrointestinal-based diseases.

Shigella IpaH9.8 limits GBP1-dependent LPS release from intracytosolic bacteria to suppress caspase-4 activation.

Pyroptosis is an inflammatory form of cell death induced upon recognition of invading microbes. During an infection, pyroptosis is enhanced in interferon-gamma-exposed cells via the actions of members of the guanylate-binding protein (GBP) family. GBPs promote caspase-4 (CASP4) activation by enhancing its interactions with lipopolysaccharide (LPS), a component of the outer envelope of Gram-negative bacteria. Once activated, CASP4 promotes the formation of noncanonical inflammasomes, signaling platforms that mediate pyroptosis. To establish an infection, intracellular bacterial pathogens, like Shigella species, inhibit pyroptosis. The pathogenesis of Shigella is dependent on its type III secretion system, which injects ~30 effector proteins into host cells. Upon entry into host cells, Shigella are encapsulated by GBP1, followed by GBP2, GBP3, GBP4, and in some cases, CASP4. It has been proposed that the recruitment of CASP4 to bacteria leads to its activation. Here, we demonstrate that two Shigella effectors, OspC3 and IpaH9.8, cooperate to inhibit CASP4-mediated pyroptosis. We show that in the absence of OspC3, an inhibitor of CASP4, IpaH9.8 inhibits pyroptosis via its known degradation of GBPs. We find that, while some LPS is present within the host cell cytosol of epithelial cells infected with wild-type Shigella, in the absence of IpaH9.8, increased amounts are shed in a GBP1-dependent manner. Furthermore, we find that additional IpaH9.8 targets, likely GBPs, promote CASP4 activation, even in the absence of GBP1. These observations suggest that by boosting LPS release, GBP1 provides CASP4-enhanced access to cytosolic LPS, thus promoting host cell death via pyroptosis.

LPS-aggregating proteins GBP1 and GBP2 are each sufficient to enhance caspase-4 activation both in cellulo and in vitro.

The gamma-interferon (IFNγ)-inducible guanylate-binding proteins (GBPs) promote host defense against gram-negative cytosolic bacteria in part through the induction of an inflammatory cell death pathway called pyroptosis. To activate pyroptosis, GBPs facilitate sensing of the gram-negative bacterial outer membrane component lipopolysaccharide (LPS) by the noncanonical caspase-4 inflammasome. There are seven human GBP paralogs, and it is unclear how each GBP contributes to LPS sensing and pyroptosis induction. GBP1 forms a multimeric microcapsule on the surface of cytosolic bacteria through direct interactions with LPS. The GBP1 microcapsule recruits caspase-4 to bacteria, a process deemed essential for caspase-4 activation. In contrast to GBP1, closely related paralog GBP2 is unable to bind bacteria on its own but requires GBP1 for direct bacterial binding. Unexpectedly, we find that GBP2 overexpression can restore gram-negative-induced pyroptosis in GBP1KO cells, without GBP2 binding to the bacterial surface. A mutant of GBP1 that lacks the triple arginine motif required for microcapsule formation also rescues pyroptosis in GBP1KO cells, showing that binding to bacteria is dispensable for GBPs to promote pyroptosis. Instead, we find that GBP2, like GBP1, directly binds and aggregates "free" LPS through protein polymerization. We demonstrate that supplementation of either recombinant polymerized GBP1 or GBP2 to an in vitro reaction is sufficient to enhance LPS-induced caspase-4 activation. This provides a revised mechanistic framework for noncanonical inflammasome activation where GBP1 or GBP2 assembles cytosol-contaminating LPS into a protein-LPS interface for caspase-4 activation as part of a coordinated host response to gram-negative bacterial infections.

A hierarchy of cell death pathways confers layered resistance to shigellosis in mice.

Bacteria of the genus Shigella cause shigellosis, a severe gastrointestinal disease driven by bacterial colonization of colonic intestinal epithelial cells. Vertebrates have evolved programmed cell death pathways that sense invasive enteric pathogens and eliminate their intracellular niche. Previously we reported that genetic removal of one such pathway, the NAIP-NLRC4 inflammasome, is sufficient to convert mice from resistant to susceptible to oral Shigella flexneri challenge (Mitchell et al., 2020). Here, we investigate the protective role of additional cell death pathways during oral mouse Shigella infection. We find that the Caspase-11 inflammasome, which senses Shigella LPS, restricts Shigella colonization of the intestinal epithelium in the absence of NAIP-NLRC4. However, this protection is limited when Shigella expresses OspC3, an effector that antagonizes Caspase-11 activity. TNFα, a cytokine that activates Caspase-8-dependent apoptosis, also provides potent protection from Shigella colonization of the intestinal epithelium when mice lack both NAIP-NLRC4 and Caspase-11. The combined genetic removal of Caspases-1, -11, and -8 renders mice hyper-susceptible to oral Shigella infection. Our findings uncover a layered hierarchy of cell death pathways that limit the ability of an invasive gastrointestinal pathogen to cause disease.

Case Report and Clinical Reasoning: Fulminant Liver Failure and Invasive Aspergillosis Following Ocrelizumab Treatment in a 21 Year-Old Woman.

We present the case of a 21 year-old woman with newly diagnosed relapsing-remitting multiple sclerosis who is given a single dose of ocrelizumab and placed on moderate-dose steroids with subsequent development of hepatic failure who goes on to develop highly fulminant systemic and central nervous system (CNS) aspergillosis. Ocrelizumab has no documented association with aspergillus infection, and moderate-dose steroids less often lead to such fulminant disease, but liver failure is associated with often-fatal aspergillus infection. We emphasize that liver failure is an underrecognized immune dysregulated state that predisposes to bacterial and fungal infections and suggest changes in diagnostic reasoning that could be considered in patients with multiple modalities of immunosuppression.

A family of conserved bacterial virulence factors dampens interferon responses by blocking calcium signaling.

Interferons (IFNs) induce an antimicrobial state, protecting tissues from infection. Many viruses inhibit IFN signaling, but whether bacterial pathogens evade IFN responses remains unclear. Here, we demonstrate that the Shigella OspC family of type-III-secreted effectors blocks IFN signaling independently of its cell death inhibitory activity. Rather, IFN inhibition was mediated by the binding of OspC1 and OspC3 to the Ca2+ sensor calmodulin (CaM), blocking CaM kinase II and downstream JAK/STAT signaling. The growth of Shigella lacking OspC1 and OspC3 was attenuated in epithelial cells and in a murine model of infection. This phenotype was rescued in both models by the depletion of IFN receptors. OspC homologs conserved in additional pathogens not only bound CaM but also inhibited IFN, suggesting a widespread virulence strategy. These findings reveal a conserved but previously undescribed molecular mechanism of IFN inhibition and demonstrate the critical role of Ca2+ and IFN targeting in bacterial pathogenesis.

The Shigella Spp. Type III Effector Protein OspB Is a Cysteine Protease.

The type III secretion system is required for virulence of many pathogenic bacteria. Bacterial effector proteins delivered into target host cells by this system modulate host signaling pathways and processes in a manner that promotes infection. Here, we define the activity of the effector protein OspB of the human pathogen Shigella spp., the etiological agent of shigellosis and bacillary dysentery. Using the yeast Saccharomyces cerevisiae as a model organism, we show that OspB sensitizes cells to inhibition of TORC1, the central regulator of growth and metabolism. In silico analyses reveal that OspB bears structural homology to bacterial cysteine proteases that target mammalian cell processes, and we define a conserved cysteine-histidine catalytic dyad required for OspB function. Using yeast genetic screens, we identify a crucial role for the arginine N-degron pathway in the yeast growth inhibition phenotype and show that inositol hexakisphosphate is an OspB cofactor. We find that a yeast substrate for OspB is the TORC1 component Tco89p, proteolytic cleavage of which generates a C-terminal fragment that is targeted for degradation via the arginine N-degron pathway; processing and degradation of Tco89p is required for the OspB phenotype. In all, we demonstrate that the Shigella T3SS effector OspB is a cysteine protease and decipher its interplay with eukaryotic cell processes. IMPORTANCEShigella spp. are important human pathogens and among the leading causes of diarrheal mortality worldwide, especially in children. Virulence depends on the Shigella type III secretion system (T3SS). Definition of the roles of the bacterial effector proteins secreted by the T3SS is key to understanding Shigella pathogenesis. The effector protein OspB contributes to a range of phenotypes during infection, yet the mechanism of action is unknown. Here, we show that S. flexneri OspB possesses cysteine protease activity in both yeast and mammalian cells, and that enzymatic activity of OspB depends on a conserved cysteine-histidine catalytic dyad. We determine how its protease activity sensitizes cells to TORC1 inhibition in yeast, finding that OspB cleaves a component of yeast TORC1, and that the degradation of the C-terminal cleavage product is responsible for OspB-mediated hypersensitivity to TORC1 inhibitors. Thus, OspB is a cysteine protease that depends on a conserved cysteine-histidine catalytic dyad.

Emerging strategies for engineering Escherichia coli Nissle 1917-based therapeutics.

Engineered microbes are rapidly being developed for the delivery of therapeutic modalities to sites of disease. Escherichia coli Nissle 1917 (EcN), a genetically tractable probiotic with a well-established human safety record, is emerging as a favored chassis. Here, we summarize the latest progress in rationally engineered variants of EcN for the treatment of infectious diseases, metabolic disorders, and inflammatory bowel diseases (IBDs) when administered orally, as well as cancers when injected directly into tumors or the systemic circulation. We also discuss emerging studies that raise potential safety concerns regarding these EcN-based strains as therapeutics due to their secretion of a genotoxic colibactin that can promote the formation of DNA double-stranded breaks in mammalian DNA.

The GBP1 microcapsule interferes with IcsA-dependent septin cage assembly around Shigella flexneri.

Many cytosolic bacterial pathogens hijack the host actin polymerization machinery to form actin tails that promote direct cell-to-cell spread, enabling these pathogens to avoid extracellular immune defenses. However, these pathogens are still susceptible to intracellular cell-autonomous immune responses that restrict bacterial actin-based motility. Two classes of cytosolic antimotility factors, septins and guanylate-binding proteins (GBPs), have recently been established to block actin tail formation by the human-adapted bacterial pathogen Shigella flexneri. Both septin cages and GBP1 microcapsules restrict S. flexneri cell-to-cell spread by blocking S. flexneri actin-based motility. While septins assemble into cage-like structures around immobile S. flexneri, GBP1 forms microcapsules around both motile and immobile bacteria. The interplay between these two defense programs remains elusive. Here, we demonstrate that GBP1 microcapsules block septin cage assembly, likely by interfering with the function of S. flexneri IcsA, the outer membrane protein that promotes actin-based motility, as this protein is required for septin cage formation. However, S. flexneri that escape from GBP1 microcapsules via the activity of IpaH9.8, a type III secreted effector that promotes the degradation of GBPs, are often captured within septin cages. Thus, our studies reveal how septin cages and GBP1 microcapsules represent complementary host cell antimotility strategies.

NAIP-NLRC4-deficient mice are susceptible to shigellosis.

Bacteria of the genus Shigella cause shigellosis, a severe gastrointestinal disease that is a major cause of diarrhea-associated mortality in humans. Mice are highly resistant to Shigella and the lack of a tractable physiological model of shigellosis has impeded our understanding of this important human disease. Here, we propose that the differential susceptibility of mice and humans to Shigella is due to mouse-specific activation of the NAIP-NLRC4 inflammasome. We find that NAIP-NLRC4-deficient mice are highly susceptible to oral Shigella infection and recapitulate the clinical features of human shigellosis. Although inflammasomes are generally thought to promote Shigella pathogenesis, we instead demonstrate that intestinal epithelial cell (IEC)-specific NAIP-NLRC4 activity is sufficient to protect mice from shigellosis. In addition to describing a new mouse model of shigellosis, our results suggest that the lack of an inflammasome response in IECs may help explain the susceptibility of humans to shigellosis.

Direct binding of polymeric GBP1 to LPS disrupts bacterial cell envelope functions.

In the outer membrane of gram-negative bacteria, O-antigen segments of lipopolysaccharide (LPS) form a chemomechanical barrier, whereas lipid A moieties anchor LPS molecules. Upon infection, human guanylate binding protein-1 (hGBP1) colocalizes with intracellular gram-negative bacterial pathogens, facilitates bacterial killing, promotes activation of the lipid A sensor caspase-4, and blocks actin-driven dissemination of the enteric pathogen Shigella. The underlying molecular mechanism for hGBP1's diverse antimicrobial functions is unknown. Here, we demonstrate that hGBP1 binds directly to LPS and induces "detergent-like" LPS clustering through protein polymerization. Binding of polymerizing hGBP1 to the bacterial surface disrupts the O-antigen barrier, thereby unmasking lipid A, eliciting caspase-4 recruitment, enhancing antibacterial activity of polymyxin B, and blocking the function of the Shigella outer membrane actin motility factor IcsA. These findings characterize hGBP1 as an LPS-binding surfactant that destabilizes the rigidity of the outer membrane to exert pleiotropic effects on the functionality of gram-negative bacterial cell envelopes.

Functional degradation: A mechanism of NLRP1 inflammasome activation by diverse pathogen enzymes.

Inflammasomes are multiprotein platforms that initiate innate immunity by recruitment and activation of caspase-1. The NLRP1B inflammasome is activated upon direct cleavage by the anthrax lethal toxin protease. However, the mechanism by which cleavage results in NLRP1B activation is unknown. In this study, we find that cleavage results in proteasome-mediated degradation of the amino-terminal domains of NLRP1B, liberating a carboxyl-terminal fragment that is a potent caspase-1 activator. Proteasome-mediated degradation of NLRP1B is both necessary and sufficient for NLRP1B activation. Consistent with our functional degradation model, we identify IpaH7.8, a Shigella flexneri ubiquitin ligase secreted effector, as an enzyme that induces NLRP1B degradation and activation. Our results provide a unified mechanism for NLRP1B activation by diverse pathogen-encoded enzymatic activities.

INAVA-ARNO complexes bridge mucosal barrier function with inflammatory signaling.

Homeostasis at mucosal surfaces requires cross-talk between the environment and barrier epithelial cells. Disruption of barrier function typifies mucosal disease. Here we elucidate a bifunctional role in coordinating this cross-talk for the inflammatory bowel disease risk-gene INAVA. Both activities require INAVA's DUF3338 domain (renamed CUPID). CUPID stably binds the cytohesin ARF-GEF ARNO to effect lateral membrane F-actin assembly underlying cell-cell junctions and barrier function. Unexpectedly, when bound to CUPID, ARNO affects F-actin dynamics in the absence of its canonical activity as a guanine nucleotide-exchange factor. Upon exposure to IL-1ß, INAVA relocates to form cytosolic puncta, where CUPID amplifies TRAF6-dependent polyubiquitination and inflammatory signaling. In this case, ARNO binding to CUPID negatively-regulates polyubiquitination and the inflammatory response. INAVA and ARNO act similarly in primary human macrophages responding to IL-1ß and to NOD2 agonists. Thus, INAVA-CUPID exhibits dual functions, coordinated directly by ARNO, that bridge epithelial barrier function with extracellular signals and inflammation.

Host-Pathogen Interactions: What the EHEC Are We Learning from Host Genome-Wide Screens?

Several genome-wide screens have been conducted to identify host cell factors involved in the pathogenesis of bacterial pathogens whose virulence is dependent on type III secretion systems (T3SSs), nanomachines responsible for the translocation of proteins into host cells. In the most recent of these, Pacheco et al. (mBio 9:e01003-18, 2018, http://mbio.asm.org/content/9/3/e01003-18.full) screened a genome-wide CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats with Cas9) knockout library for host proteins involved in the pathogenesis of enterohemorrhagic Escherichia coli (EHEC). Their study revealed an unrecognized link between EHEC's two major virulence determinants (its T3SS and Shiga toxins). We discuss these findings in light of data from three other genome-wide screens. Each of these studies uncovered multiple host cell determinants, which curiously share little to no overlap but primarily are involved in mediating early interactions between T3SSs and host cells. We therefore consider how each screen was performed, the advantages and disadvantages of each, and how follow-up studies might be designed to address these issues.