Development and validation of systems for genetic manipulation of the Old World tick-borne relapsing fever spirochete, Borrelia duttonii.

Relapsing fever (RF), a vector-borne disease caused by Borrelia spp., is characterized by recurring febrile episodes due to repeated bouts of bacteremia. RF spirochetes can be geographically and phylogenetically divided into two distinct groups; Old World RF Borrelia (found in Africa, Asia, and Europe) and New World RF Borrelia (found in the Americas). While RF is a rarely reported disease in the Americas, RF is prevalent in endemic parts of Africa. Despite phylogenetic differences between Old World and New World RF Borrelia and higher incidence of disease associated with Old World RF spirochete infection, genetic manipulation has only been described in New World RF bacteria. Herein, we report the generation of genetic tools for use in the Old World RF spirochete, Borrelia duttonii. We describe methods for transformation and establish shuttle vector- and integration-based approaches for genetic complementation, creating green fluorescent protein (gfp)-expressing B. duttonii strains as a proof of principle. Allelic exchange mutagenesis was also used to inactivate a homolog of the Borrelia burgdorferi p66 gene, which encodes an important virulence factor, in B. duttonii and demonstrate that this mutant was attenuated in a murine model of RF. Finally, the B. duttonii p66 mutant was complemented using shuttle vector- and cis integration-based approaches. As expected, complemented p66 mutant strains were fully infectious, confirming that P66 is required for optimal mammalian infection. The genetic tools and techniques reported herein represent an important advancement in the study of RF Borrelia that allows for future characterization of virulence determinants and colonization factors important for the enzootic cycle of Old World RF spirochetes.

MicroRNAs Contribute to Host Response to Coxiella burnetii.

MicroRNAs (miRNAs), a class of small noncoding RNAs, are critical to gene regulation in eukaryotes. They are involved in modulating a variety of physiological processes, including the host response to intracellular infections. Little is known about miRNA functions during infection by Coxiella burnetii, the causative agent of human Q fever. This bacterial pathogen establishes a large replicative vacuole within macrophages by manipulating host processes such as apoptosis and autophagy. We investigated miRNA expression in C. burnetii-infected macrophages and identified several miRNAs that were down- or upregulated during infection. We further explored the functions of miR-143-3p, an miRNA whose expression is downregulated in macrophages infected with C. burnetii, and show that increasing the abundance of this miRNA in human cells results in increased apoptosis and reduced autophagy-conditions that are unfavorable to C. burnetii intracellular growth. In sum, this study demonstrates that C. burnetii infection elicits a robust miRNA-based host response, and because miR-143-3p promotes apoptosis and inhibits autophagy, downregulation of miR-143-3p expression during C. burnetii infection likely benefits the pathogen.

Neurotransmitter System-Targeting Drugs Antagonize Growth of the Q Fever Agent, Coxiella burnetii, in Human Cells.

Coxiella burnetii is a highly infectious, intracellular, Gram-negative bacterial pathogen that causes human Q fever, an acute flu-like illness that can progress to chronic endocarditis. C. burnetii is transmitted to humans via aerosols and has long been considered a potential biological warfare agent. Although antibiotics, such as doxycycline, effectively treat acute Q fever, a recently identified antibiotic-resistant strain demonstrates the ability of C. burnetii to resist traditional antimicrobials, and chronic disease is extremely difficult to treat with current options. These findings highlight the need for new Q fever therapeutics, and repurposed drugs that target eukaryotic functions to prevent bacterial replication are of increasing interest in infectious disease. To identify this class of anti-C. burnetii therapeutics, we screened a library of 727 FDA-approved or late-stage clinical trial compounds using a human macrophage-like cell model of infection. Eighty-eight compounds inhibited bacterial replication, including known antibiotics, antipsychotic or antidepressant treatments, antihistamines, and several additional compounds used to treat a variety of conditions. The majority of identified anti-C. burnetii compounds target host neurotransmitter system components. Serotoninergic, dopaminergic, and adrenergic components are among the most highly represented targets and potentially regulate macrophage activation, cytokine production, and autophagy. Overall, our screen identified multiple host-directed compounds that can be pursued for potential use as anti-C. burnetii drugs. IMPORTANCE Coxiella burnetii causes the debilitating disease Q fever in humans. This infection is difficult to treat with current antibiotics and can progress to long-term, potentially fatal infection in immunocompromised individuals or when treatment is delayed. Here, we identified many new potential treatment options in the form of drugs that are either FDA approved or have been used in late-stage clinical trials and target human neurotransmitter systems. These compounds are poised for future characterization as nontraditional anti-C. burnetii therapies.

Coxiella burnetii Requires Host Eukaryotic Initiation Factor 2α Activity for Efficient Intracellular Replication.

Coxiella burnetii is the causative agent of human Q fever, eliciting symptoms that range from acute fever and fatigue to chronic fatal endocarditis. C. burnetii is a Gram-negative intracellular bacterium that replicates within an acidic lysosome-like parasitophorous vacuole (PV) in human macrophages. During intracellular growth, C. burnetii delivers bacterial proteins directly into the host cytoplasm using a Dot/Icm type IV secretion system (T4SS). Multiple T4SS effectors localize to and/or disrupt the endoplasmic reticulum (ER) and secretory transport, but their role in infection is unknown. During microbial infection, unfolded nascent proteins may exceed the folding capacity of the ER, activating the unfolded protein response (UPR) and restoring the ER to its normal physiological state. A subset of intracellular pathogens manipulates the UPR to promote survival and replication in host cells. In this study, we investigated the impact of C. burnetii infection on activation of the three arms of the UPR. An inhibitor of the UPR antagonized PV expansion in macrophages, indicating this process is needed for bacterial replication niche formation. Protein kinase RNA-like ER kinase (PERK) signaling was activated during infection, leading to increased levels of phosphorylated eukaryotic initiation factor α, which was required for C. burnetii growth. Increased production and nuclear translocation of the transcription factor ATF4 also occurred, which normally drives expression of the proapoptotic C/EBP homologous protein (CHOP). CHOP protein production increased during infection; however, C. burnetii actively prevented CHOP nuclear translocation and downstream apoptosis in a T4SS-dependent manner. The results collectively demonstrate interplay between C. burnetii and specific components of the eIF2α signaling cascade to parasitize human macrophages.

Infection of Primary Human Alveolar Macrophages Alters Staphylococcus aureus Toxin Production and Activity.

Pulmonary pathogens encounter numerous insults, including phagocytic cells designed to degrade bacteria, while establishing infection in the human lung. Staphylococcus aureus is a versatile, opportunistic pathogen that can cause severe pneumonia, and methicillin-resistant isolates are of particular concern. Recent reports present conflicting data regarding the ability of S. aureus to survive and replicate within macrophages. However, due to use of multiple strains and macrophage sources, making comparisons between reports remains difficult. Here, we established a disease-relevant platform to study innate interactions between S. aureus and human lungs. Human precision-cut lung slices (hPCLS) were subjected to infection by S. aureus LAC (methicillin-resistant) or UAMS-1 (methicillin-sensitive) isolates. Additionally, primary human alveolar macrophages (hAMs) were infected with S. aureus, and antibacterial activity was assessed. Although both S. aureus isolates survived within hAM phagosomes, neither strain replicated efficiently in these cells. S. aureus was prevalent within the epithelial and interstitial regions of hPCLS, with limited numbers present in a subset of hAMs, suggesting that the pathogen may not target phagocytic cells for intracellular growth during natural pulmonary infection. S. aureus-infected hAMs mounted a robust inflammatory response that reflected natural human disease. S. aureus LAC was significantly more cytotoxic to hAMs than UAMS-1, potentially due to isolate-specific virulence factors. The bicomponent toxin Panton-Valentine leukocidin was not produced during intracellular infection, while alpha-hemolysin was produced but was not hemolytic, suggesting that hAMs alter toxin activity. Overall, this study defined a new disease-relevant infection platform to study S. aureus interaction with human lungs and to define virulence factors that incapacitate pulmonary cells.

Functional inhibition of acid sphingomyelinase disrupts infection by intracellular bacterial pathogens.

Intracellular bacteria that live in host cell-derived vacuoles are significant causes of human disease. Parasitism of low-density lipoprotein (LDL) cholesterol is essential for many vacuole-adapted bacteria. Acid sphingomyelinase (ASM) influences LDL cholesterol egress from the lysosome. Using functional inhibitors of ASM (FIASMAs), we show that ASM activity is key for infection cycles of vacuole-adapted bacteria that target cholesterol trafficking-Anaplasma phagocytophilum, Coxiella burnetii, Chlamydia trachomatis, and Chlamydia pneumoniae. Vacuole maturation, replication, and infectious progeny generation by A. phagocytophilum, which exclusively hijacks LDL cholesterol, are halted and C. burnetii, for which lysosomal cholesterol accumulation is bactericidal, is killed by FIASMAs. Infection cycles of Chlamydiae, which hijack LDL cholesterol and other lipid sources, are suppressed but less so than A. phagocytophilum or C. burnetii A. phagocytophilum fails to productively infect ASM-/- or FIASMA-treated mice. These findings establish the importance of ASM for infection by intracellular bacteria and identify FIASMAs as potential host-directed therapies for diseases caused by pathogens that manipulate LDL cholesterol.