Abby R. Kroken

Pseudomonas aeruginosa is a high-priority pathogen and significant burden to health care worldwide. Although often regarded as an extracellular pathogen, P. aeruginosa is also capable of existing intracellularly in multiple cell types, including epithelial cells, goblet cells, and macrophages. This designation is attributed to two of its type three secretion system (T3SS)–dependent exotoxins, ExoS and ExoT, which inactivate host proteins that facilitate phagocytosis. However, studies investigating intracellular bacteria show that ExoS can paradoxically facilitate survival and replication, seemingly overriding the anti-internalization properties of itself and ExoT through its ADP-ribosylation activity. Here, we set out to define the individual and combined contributions of ExoS and ExoT by examining how each of their two functional domain activities affects epithelial cell invasion. Through in vitro biochemical assays, we found that ExoS is capable of ADP-ribosylating ExoT and their shared human cofactor, 14-3-3. We also found that ExoT ADP-ribosylates itself, unexpectedly enhancing its GTPase-activating protein (GAP) activity. Using fluorescence microscopy, we found that GAP activity of either exotoxin does not block internalization events, but is instead associated with fewer internalized bacteria entering a phase of rapid cytoplasmic replication. We demonstrate that ExoS ADP-ribosyltransferase activity is positively associated with vacuolar exit and cytoplasmic replication, and delivery of ExoS from extracellular bacteria triggers the intracellular T3SS⁻ subpopulation to become T3SS ⁺ and exit the vacuole. Overall, this study highlights the underappreciated ability of P. aeruginosa to become intracellular and delineates how the enzymatic domains of ExoS and ExoT dictate the intracellular localization of the pathogen.

Pseudomonas aeruginosa is known to infect both phagocytic and non-phagocytic host cells, where it exhibits a complex intracellular lifestyle strategy for host survival that is governed by the expression and regulation of its type III secretion system (T3SS). To better understand such complexities in the context of niche bacterial populations in P. aeruginosa and other gram-negatives, here we construct and characterize a novel and versatile dual-reporter plasmid, C onstitutive G FP, V ariable mS carlet-I (pCG-VmS), whose utility proves useful for longitudinal assays of transcriptional reporter activity in vitro , ex vivo , and in whole animals. As an added utility, we show that pCG-VmS can also be used for translational fusions to monitor bacterial cellular protein localization. We then exploit pCG-VmS to visualize P. aeruginosa T3SS expression in a human corneal cell line that identifies a distinct intracellular “quiescent” population, where the T3SS is never expressed. An ex vivo murine eye infection model is then used to verify the existence of the two ‘T3SS-on” versus ‘T3SS-off’ expression profiles in infected tissues. In sum, pCG-VmS allows for novel spatiotemporal insights into P. aeruginosa protein localization and infection routes and can be tailored to other plasmid backbones amenable to different gram-negative bacteria. IMPORTANCE: In this study, we develop a novel, well-optimized fluorescent dual-reporter system, pCG-VmS, which can be used for both transcriptional monitoring and protein localization. We demonstrate its use with a range of in vitro and in vivo experiments with Pseudomonas aeruginosa and whole-animal imaging of squid colonization with Vibrio fischeri . After showing pCG-VmS efficacy and versatility, we use the plasmid to simultaneously visualize the naturally occurring type-3 secretion system (T3SS) active (T3SS-on) and “quiescent” (T3SS-off) populations during intracellular infection of a human corneal epithelial cell line and the murine eye. Taken together, pCG-VmS is well-balanced in its optical traits and can be used for a variety of both in vitro and in vivo studies to help separate and further interrogate difficult niche spatiotemporal bacterial populations.

Streptococcus pneumoniae (the pneumococcus) is a human bacterial pathogen and the major cause of bacterial pneumonia, which can further develop into sepsis. The pneumococcus has evolved over 107 antigenically-distinct serotypes that are defined by the chemical composition of its capsule; its major virulence factor and the main protective antigen within the vaccine. Owing to its capsule diversification, certain serotypes are known for being more effective at either colonization or invasiveness. It has historically been accepted that these differences are due to the chemical properties of the capsule itself, whose biosynthetic genes are encoded in a single capsular polysaccharide (cps) operon. Here we show that infection outcomes can also be the result of a serotype’s subtle, natural variations within the regulatory region which results in unique transcriptional control of the cps locus. We propose that these data explain, in part, how this pathogen might rapidly evolve its capsule expression to accommodate evolutionary pressures introduced from pneumococcal vaccines, without having to rely on genetic recombination.

The universally conserved α-oxoaldehydes glyoxal (GO) and methylglyoxal (MGO) are toxic metabolic byproducts whose accumulation can lead to cell death. In the absence of a known, natural inducer of the GO-specific response in prokaryotes, we exploited RNA-seq to define a GO response in the bacterial pathogen Pseudomonas aeruginosa. The highest upregulated operon consisted of the known glyoxalase (gloA2) and an antibiotic monooxygenase (ABM) domain of unknown function - renamed here A ldehyde r esponsive q uorum-sensing I nhibitor (ArqI). The arqI-gloA2 operon is highly specific to GO induction and ArqI protein responds by migrating to the flagellar pole. An ArqI atomic structure revealed several unique features to the ABM family, including a ‘pinwheel’ hexamer harboring a GO-derived post-translational modification on a conserved arginine residue (Arg49). Induction of ArqI abrogates production of the Pseudomonas Quinolone Signal (PQS) quorum sensing molecule and was found to directly interact with PqsA; the first enzyme in the PQS biosynthesis pathway. Finally, we use a sepsis model of infection to reveal a survival requirement for arqI-gloA2 in blood-rich organs (heart, spleen, liver and lung). Here we define a global GO response in a pathogen, identify and characterize the first GO-specific operon and implicate its role in PQS production and host survival.

Bacterial dispersal from a biofilm is presently the least-studied step of the biofilm life cycle. The symbiotic bacterial species Vibrio fischeri is a model organism for studying biofilms relevant to a eukaryotic host; however, methodology is lacking to readily study the dispersal of this microbe from biofilms formed in the lab. Here, we adapted a time-lapse assay to visualize biofilm dispersal by V. fischeri . We observed biofilm formation and dispersal for multiple V. fischeri isolates, which displayed a variety of biofilm architecture phenotypes and dispersal dynamics. We then investigated V. fischeri strain ES114 using genetic tools and mutants available for this strain. ES114 exhibited calcium-dependent biofilm formation followed by a rapid (less than 10 min) coordinated dispersal event that occurred approximately 5 h from the experimental start. Biofilm dispersal was largely independent of the dispersal-promoting protease encoded by lapG . Although we found no role under our conditions for either biofilm formation or dispersal for several other factors including polysaccharides and autoinducers, we determined that biofilm formation was enhanced, and dispersal was delayed, with increased concentrations of calcium. Furthermore, biofilm formation depended on the calcium-responsive diguanylate cyclase (DGC) CasA, and dispersal could be modulated by overexpressing CasA. Our work has thus developed a new tool for the V. fischeri field and uncovered a key role for calcium signaling and c-di-GMP in early biofilm formation and dispersal in V. fischeri . IMPORTANCE: Biofilm formation and dispersal are critical steps in both symbiotic and pathogenic colonization. Relative to biofilm formation, the process of dispersal in the model symbiont Vibrio fischeri , and other bacteria, is understudied. Here, we adapted an imaging assay to study early biofilm formation and the dispersal process in V. fischeri . We demonstrated that our assay can quantify biofilm formation and dispersal over time, can reveal phenotypic differences in diverse natural wild-type isolates, and is sensitive enough to investigate the impact of environmental factors. Our data confirm that calcium is a potent biofilm formation signal and identify the diguanylate cyclase CasA as a key regulator. This work leads the way for more in-depth research about unknown mechanisms of biofilm dispersal.

Pseudomonas aeruginosa is a significant cause of global morbidity and mortality. Although it is often regarded as an extracellular pathogen toward human cells, numerous investigations report its ability to survive and replicate within host cells, and additional studies demonstrate specific mechanisms enabling it to adopt an intracellular lifestyle. This ability of P. aeruginosa remains less well-investigated than that of other intracellular bacteria, although it is currently gaining attention. If intracellular bacteria are not killed after entering host cells, they may instead receive protection from immune recognition and experience reduced exposure to antibiotic therapy, among additional potential advantages shared with other facultative intracellular pathogens. For this review, we compiled studies that observe intracellular P. aeruginosa across strains, cell types, and experimental systems in vitro , as well as contextualize these findings with the few studies that report similar observations in vivo . We also seek to address key findings that drove the perception that P. aeruginosa remains extracellular in order to reconcile what is currently understood about intracellular pathogenesis and highlight open questions regarding its contribution to disease.

Pathogenesis of Pseudomonas aeruginosa infections can include bacterial survival inside epithelial cells. Previously, we showed that this involves multiple roles played by the type three secretion system (T3SS), and specifically the effector ExoS. This includes ExoS-dependent inhibition of a lytic host cell response that subsequently enables intracellular replication. Here, we studied the underlying cell death response to intracellular P. aeruginosa , comparing wild-type to T3SS mutants varying in capacity to induce cell death and that localize to different intracellular compartments. Results showed that corneal epithelial cell death induced by intracellular P. aeruginosa lacking the T3SS, which remains in vacuoles, correlated with the activation of nuclear factor-κB as measured by p65 relocalization and tumor necrosis factor alpha transcription and secretion. Deletion of caspase-4 through CRISPR-Cas9 mutagenesis delayed cell death caused by these intracellular T3SS mutants. Caspase-4 deletion also countered more rapid cell death caused by T3SS effector-null mutants still expressing the T3SS apparatus that traffic to the host cell cytoplasm, and in doing so rescued intracellular replication normally dependent on ExoS. While HeLa cells lacked a lytic death response to T3SS mutants, it was found to be enabled by interferon gamma treatment. Together, these results show that epithelial cells can activate the noncanonical inflammasome pathway to limit proliferation of intracellular P. aeruginosa , not fully dependent on bacterially driven vacuole escape. Since ExoS inhibits the lytic response, the data implicate targeting of caspase-4, an intracellular pattern recognition receptor, as another contributor to the role of ExoS in the intracellular lifestyle of P. aeruginosa . IMPORTANCE: Pseudomonas aeruginosa can exhibit an intracellular lifestyle within epithelial cells in vivo and in vitro . The type three secretion system (T3SS) effector ExoS contributes via multiple mechanisms, including extending the life of invaded host cells. Here, we aimed to understand the underlying cell death inhibited by ExoS when P. aeruginosa is intracellular. Results showed that intracellular P. aeruginosa lacking T3SS effectors could elicit rapid cell lysis via the noncanonical inflammasome pathway. Caspase-4 contributed to cell lysis even when the intracellular bacteria lacked the entire T33S and were consequently unable to escape vacuoles, representing a naturally occurring subpopulation during wild-type infection. Together, the data show the caspase-4 inflammasome as an epithelial cell defense against intracellular P. aeruginosa , and implicate its targeting as another mechanism by which ExoS preserves the host cell replicative niche.

Pathogenesis of Pseudomonas aeruginosa infections can include bacterial survival inside epithelial cells. Previously, we showed this involves multiple roles played by the type three-secretion system (T3SS), and specifically the effector ExoS. This includes ExoS-dependent inhibition of a lytic host cell response that subsequently enables intracellular replication. Here, we studied the underlying cell death response to intracellular P. aeruginosa , comparing wild-type to T3SS mutants varying in capacity to induce cell death and that localize to different intracellular compartments. Results showed that corneal epithelial cell death induced by intracellular P. aeruginosa lacking the T3SS, which remains in vacuoles, correlated with activation of NF-κB as measured by p65 relocalization and TNFα transcription and secretion. Deletion of caspase-4 through CRISPR-Cas9 mutagenesis delayed cell death caused by these intracellular T3SS mutants. Caspase-4 deletion also countered more rapid cell death caused by T3SS effector-null mutants still expressing the TSSS apparatus that traffic to the host cell cytoplasm, and in doing so rescued intracellular replication normally dependent on ExoS. While HeLa cells lacked a lytic death response to T3SS mutants, it was found to be enabled by interferon gamma treatment. Together, these results show that epithelial cells can activate the noncanonical inflammasome pathway to limit proliferation of intracellular P. aeruginosa , not fully dependent on bacterially-driven vacuole escape. Since ExoS inhibits the lytic response, the data implicate targeting of caspase-4, an intracellular pattern recognition receptor, as another contributor to the role of ExoS in the intracellular lifestyle of P. aeruginosa . Importance: Pseudomonas aeruginosa can exhibit an intracellular lifestyle within epithelial cells in vivo and in vitro. The type three secretion system (T3SS) effector ExoS contributes via multiple mechanisms, including extending the life of invaded host cells. Here, we aimed to understand the underlying cell death inhibited by ExoS when P. aeruginosa is intracellular. Results showed that intracellular P. aeruginosa lacking T3SS effectors could elicit rapid cell lysis via the non-canonical inflammasome pathway. Caspase-4 contributed to cell lysis even when the intracellular bacteria lacked the entire T33S and were consequently unable to escape vacuoles, representing a naturally occurring subpopulation during wildtype infection. Together, the data show the caspase-4 inflammasome as an epithelial cell defense against intracellular P. aeruginosa , and implicate its targeting as another mechanism by which ExoS preserves the host cell replicative niche.

Host cell invasion can contribute to disease pathogenesis by the opportunistic pathogen Pseudomonas aeruginosa . Previously, we showed that the type III secretion system (T3SS) of invasive P. aeruginosa strains modulates cell entry and subsequent escape from vacuolar trafficking to host lysosomes. However, we also showed that mutants lacking either type IV pili (T4P) or T4P-dependent twitching motility (i) were defective in traversing cell multilayers, (ii) caused less pathology in vivo , and (iii) had a reduced capacity to exit invaded cells. Here, we report that after vacuolar escape, intracellular P. aeruginosa can use T4P-dependent twitching motility to disseminate throughout the host cell cytoplasm. We further show that this strategy for intracellular dissemination does not depend on flagellin and resists both host actin and host microtubule disruption. This differs from mechanisms used by previously studied pathogens that utilize either host actin or microtubules for intracellular dissemination independently of microbe motility appendages.