Many bacterial pathogens employ multicomponent protein complexes to deliver macromolecules directly into their eukaryotic host cell to promote infection. Some Gram-negative pathogens use a versatile type IV secretion system (T4SS) that can translocate DNA or proteins into host cells. T4SSs represent major bacterial virulence determinants and have recently been the focus of intense research efforts designed to better understand and combat infectious diseases. Interestingly, although the two major classes of T4SSs function in a similar manner to secrete proteins, the translocated “effectors” vary substantially from one organism to another. In fact, differing effector repertoires likely contribute to organism-specific host cell interactions and disease outcomes. In this review, we discuss the current state of T4SS research, with an emphasis on intracellular bacterial pathogens of humans and the diverse array of translocated effectors used to manipulate host cells.

Summary The intracellular bacterial pathogen Coxiella burnetii is a category B select agent that causes human Q fever. In vivo, C. burnetii targets alveolar macrophages wherein the pathogen replicates in a lysosome-like parasitophorous vacuole (PV). In vitro, C. burnetii infects a variety of cultured cell lines that have collectively been used to model the pathogen’s infectious cycle. However, differences in the cellular response to infection have been observed, and virulent C. burnetii isolate infection of host cells has not been well defined. Because alveolar macrophages are routinely implicated in disease, we established primary human alveolar macrophages (hAMs) as an in vitro model of C. burnetii-host cell interactions. C. burnetii pathotypes, including acute disease and endocarditis isolates, replicated in hAMs, albeit with unique PV properties. Each isolate replicated in large, typical PV and small, non-fused vacuoles, and lipid droplets were present in avirulent C. burnetii PV. Interestingly, a subset of small vacuoles harbored single organisms undergoing degradation. Prototypical PV formation and bacterial growth in hAMs required a functional type IV secretion system, indicating C. burnetii secretes effector proteins that control macrophage functions. Avirulent C. burnetii promoted sustained activation of Akt and Erk1/2 pro-survival kinases and short term phosphorylation of stress-related p38. Avirulent organisms also triggered a robust, early pro-inflammatory response characterized by increased secretion of TNF-α and IL-6, while virulent isolates elicited substantially reduced secretion of these cytokines. A corresponding increase in pro- and mature IL-1β occurred in hAMs infected with avirulent C. burnetii, while little accumulation was observed following infection with virulent isolates. Finally, treatment of hAMs with IFN-γ controlled intracellular replication, supporting a role for this antibacterial insult in the host response to C. burnetii. Collectively, the current results demonstrate the hAM model is a human disease-relevant platform for defining novel innate immune responses to C. burnetii.

cCoxiella burnetii is an intracellular Gram-negative bacterium that causes human Q fever, a flu-like disease that can progress to chronic, life-threatening endocarditis. In humans, C. burnetii infects alveolar macrophages and promotes phagosomal fusion with autophagosomes and lysosomes, establishing a unique parasitophorous vacuole (PV) in which to replicate. The pathogen uses a Dot/Icm type IV secretion system (T4SS) to deliver effector proteins to the host cytoplasm, where they alter cellular processes to benefit the pathogen. The T4SS is required for PV expansion and prevention of apoptosis, but little else is known about the role of the system during intracellular growth. Recent reports suggest that C. burnetii actively recruits autophagosomes to the PV to deliver nutrients to the pathogen and provide membrane for the expanding vacuole. In this study, we examined the role of the T4SS in mediating PV interactions with autophagosomes. We found that the autophagy-related proteins LC3 and p62 localized to wild-type PV but not to T4SS mutant organism-containing phagosomes in human macrophage-like cells, primary human alveolar macrophages, and Chinese hamster ovary cells. However, while lipidated LC3 levels were elevated regardless of T4SS activity, no p62 turnover was observed during C. burnetii growth in macrophages, suggesting that the pathogen recruits preformed autophagosomes. When the T4SS was activated 24 h after infection, autophagosome recruitment ensued, indicating that autophagosome interactions are dispensable for initial PV maturation to a phagolysosome-like compartment but are involved in vacuole expansion. Together, these results demonstrate that C. burnetii actively directs PV-autophagosome interactions by using the Dot/Icm T4SS.

The intracellular bacterial agent of Q fever, Coxiella burnetii, translocates effector proteins into its host cell cytosol via a Dot/ Icm type IV secretion system (T4SS). The T4SS is essential for parasitophorous vacuole formation, intracellular replication, and inhibition of host cell death, but the effectors mediating these events remain largely undefined. Six Dot/Icm substrate-encoding genes were recently discovered on the C. burnetii cryptic QpH1 plasmid, three of which are conserved among all C. burnetii isolates, suggesting that they are critical for conserved pathogen functions. However, the remaining hypothetical proteins encoded by plasmid genes have not been assessed for their potential as T4SS substrates. In the current study, we further defined the T4SS effector repertoire encoded by the C. burnetii QpH1, QpRS, and QpDG plasmids that were originally isolated from acute-disease, chronic-disease, and severely attenuated isolates, respectively. Hypothetical proteins, including those specific to QpRS or QpDG, were screened for translocation using the well-established Legionella pneumophila T4SS secretion model. In total, six novel plasmid-encoded proteins were translocated into macrophage-like cells by the Dot/Icm T4SS. Four newly identified effectors are encoded by genes present only on the QpDG plasmid from severely attenuated Dugway isolates, suggesting that the presence of specific effectors correlates with decreased virulence. These results further support the idea of a critical role for extrachromosomal elements in C. burnetii pathogenesis.

fIn Staphylococcus aureus, metabolism is intimately linked with virulence determinant biosynthesis, and several metabolite-responsive regulators have been reported to mediate this linkage. S. aureus possesses at least three members of the RpiR family of transcriptional regulators. Of the three RpiR homologs, RpiRc is a potential regulator of the pentose phosphate pathway, which also regulates RNAIII levels. RNAIII is the regulatory RNA of the agr quorum-sensing system that controls virulence determinant synthesis. The effect of RpiRc on RNAIII likely involves other regulators, as the regulators that bind the RNAIII promoter have been intensely studied. To determine which regulators might bridge the gap between RpiRc and RNAIII, sarA, sigB, mgrA, and acnA mutations were introduced into an rpiRc mutant background, and the effects on RNAIII were determined. Additionally, phenotypic and genotypic differences were examined in the single and double mutant strains, and the virulence of select strains was examined using two different murine infection models. The data suggest that RpiRc affects RNAIII transcription and the synthesis of virulence determinants in concert with B , SarA, and the bacterial metabolic status to negatively affect virulence. Staphylococcus aureus is a major human pathogen causing a diverse range of infections, from superficial skin and wound infections to life-threatening diseases such as bacteremia, endocarditis, osteomyelitis, deep tissue abscesses, or pneumonia (1). The pathogenicity of S. aureus is due in part to its ability to produce a large number of virulence determinants, including secreted proteins (e.g., toxins and proteases), cell wall-associated proteins (e.g., protein A and fibronectin binding proteins), and extracellular polysaccharides (i.e., capsule and polysaccharide intercellular adhesion). During colonization and infection of a host, S. aureus must adapt to rapidly changing environmental and nutritional conditions by coordinating the transcription and translation of physiologic and virulence genes (2). To coordinately control these cellular processes, S. aureus has evolved or acquired a network of regulators such as the recently identified family of proteins, RpiR, that affect pentose phosphate pathway activity and virulence determinant synthesis (3).Central to the regulatory network is the accessory gene regulator (Agr) system, a regulator of virulence determinant synthesis that responds to the bacterial population density (4). The agr locus consists of two divergent transcriptional units, RNAII and RNAIII, driven by the P2 and P3 promoters, respectively. RNAII comprises the agrBDCA operon, of which the agrBD gene products are involved in the synthesis, transport, and maturation of an autoinducing peptide (AIP). As the cell density increases, AIP accumulates in the extracellular milieu and when a threshold is achieved the two-component system AgrCA responds by activating transcription of both P2 and P3 promoters. The P3 promoter drives transcription of RNAIII, which is the re...

Summary Intracellular bacterial pathogens often subvert apoptosis signaling to regulate survival of their host cell, allowing propagation of the bacterial population. Coxiella burnetii, the intracellular agent of human Q fever, inhibits host cell apoptosis through several mechanisms, including prevention of mitochondrial cytochrome c release, triggering of an anti-apoptotic transcriptional program, and activation of pro-survival kinases. To control host cell survival, C. burnetii delivers effector proteins to the eukaryotic cytosol using a specialized Dot/Icm type IV secretion system (T4SS). Effectors are predicted to regulate activity of pro-survival host signaling proteins, such as Akt and cAMP-dependent protein kinase (PKA), to control infection. Here, we show that host PKA activity is required for C. burnetii inhibition of macrophage apoptosis. PKA is activated during infection and inhibits activity of the pro-apoptotic protein Bad via phosphorylation. Bad is also phosphorylated at an Akt-specific residue, indicating C. burnetii uses two kinases to fully inactivate Bad. Additionally, Bad and the tethering protein 14-3-3β co-localize at the C. burnetii parasitophorous vacuole (PV) membrane during infection, an event predicted to alter Bad promotion of apoptosis. Inhibiting PKA activity prevents Bad recruitment to the PV, but the protein is retained at the membrane during induction of apoptosis. Finally, PKA regulatory subunit I (RI) traffics to the PV membrane in a T4SS-dependent manner, suggesting a C. burnetii effector(s) regulates PKA-dependent activities. This study is the first to demonstrate subversion of host PKA activity by an intracellular bacterial pathogen to prevent apoptosis and survive within macrophages.

cCoxiella burnetii is an intracellular bacterial pathogen that causes human Q fever, an acute debilitating flu-like illness that can also present as chronic endocarditis. Disease typically occurs following inhalation of contaminated aerosols, resulting in an initial pulmonary infection. In human cells, C. burnetii generates a replication niche termed the parasitophorous vacuole (PV) by directing fusion with autophagosomes and lysosomes. C. burnetii requires this lysosomal environment for replication and uses a Dot/Icm type IV secretion system to generate the large PV. However, we do not understand how C. burnetii evades the intracellular immune surveillance that triggers an inflammatory response. We recently characterized human alveolar macrophage (hAM) infection in vitro and found that avirulent C. burnetii triggers sustained interleukin-1␤ (IL-1␤) production. Here, we evaluated infection of ex vivo human lung tissue, defining a valuable approach for characterizing C. burnetii interactions with a human host. Within whole lung tissue, C. burnetii preferentially replicated in hAMs. Additionally, IL-1␤ production correlated with formation of an apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC)-dependent inflammasome in response to infection. We also assessed potential activation of a human-specific noncanonical inflammasome and found that caspase-4 and caspase-5 are processed during infection. Interestingly, although inflammasome activation is closely linked to pyroptosis, lytic cell death did not occur following C. burnetii-triggered inflammasome activation, indicating an atypical response after intracellular detection. Together, these studies provide a novel platform for studying the human innate immune response to C. burnetii. Coxiella burnetii is a Gram-negative, obligate intracellular pathogen that causes Q fever in humans (1). Acute Q fever presents with flu-like symptoms; however, infection can persist and cause life-threatening endocarditis. C. burnetii is a category B select agent due to an aerosol mode of transmission, low infectious dose, and environmental stability (2). The pathogen has a unique intracellular lifestyle that requires replication within an acidic lysosome-like parasitophorous vacuole (PV) in macrophages (3). Using a Dot/Icm type IV secretion system (T4SS) to secrete bacterial proteins into the host cytoplasm (4, 5), C. burnetii hijacks the host cell to control signaling cascades and heterotypic fusion with endosomes, autophagosomes, and lysosomes to establish the PV (6). Although C. burnetii intracellular trafficking has been well characterized, understanding of the initial interactions between the pathogen and its human host is lacking. Although small animal models provide beneficial information about the host response to C. burnetii, they do not accurately mimic the human lung response to infection, presenting the need for improved systems. For example, mouse alveolar macrophages degrade C. burnetii (7), while the pathogen replicates e...

Coxiella burnetii causes human Q fever, a zoonotic disease that presents with acute flu-like symptoms and can result in chronic life-threatening endocarditis. In human alveolar macrophages, C. burnetii uses a Dot/Icm type IV secretion system (T4SS) to generate a phagolysosome-like parasitophorous vacuole (PV) in which to replicate. The T4SS translocates effector proteins, or substrates, into the host cytosol, where they mediate critical cellular events, including interaction with autophagosomes, PV formation, and prevention of apoptosis. Over 100 C. burnetii Dot/Icm substrates have been identified, but the function of most remains undefined. Here, we identified a novel Dot/Icm substrate-encoding open reading frame (CbuD1884) present in all C. burnetii isolates except the Nine Mile reference isolate, where the gene is disrupted by a frameshift mutation, resulting in a pseudogene. The CbuD1884 protein contains two transmembrane helices (TMHs) and a coiled-coil domain predicted to mediate protein-protein interactions. The C-terminal region of the protein contains a predicted Dot/Icm translocation signal and was secreted by the T4SS, while the N-terminal portion of the protein was not secreted. When ectopically expressed in eukaryotic cells, the TMH-containing N-terminal region of the CbuD1884 protein trafficked to the endoplasmic reticulum (ER), with the C terminus dispersed nonspecifically in the host cytoplasm. This new Dot/Icm substrate is now termed ElpA (ER-localizing protein A). Full-length ElpA triggered substantial disruption of ER structure and host cell secretory transport. These results suggest that ElpA is a pathotype-specific T4SS effector that influences ER function during C. burnetii infection.