Virulent Coxiella burnetii pathotypes productively infect primary human alveolar macrophages

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 harboured 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‐termphosphorylation 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.     

Cell cycle and cell death are not necessary for appressorium formation and plant infection in the fungal plant pathogen <Emphasis Type="Italic">Colletotrichum gloeosporioides</Emphasis>

Background  

In order to initiate plant infection, fungal spores must germinate and penetrate into the host plant. Many fungal species differentiate specialized infection structures called appressoria on the host surface, which are essential for successful pathogenic development. In the model plant pathogen Magnaporthe grisea completion of mitosis and autophagy cell death of the spore are necessary for appressoria-mediated plant infection; blocking of mitosis prevents appressoria formation, and prevention of autophagy cell death results in non-functional appressoria.     

Molecular characterization of Coxiella burnetii isolates by infrequent restriction site-PCR and MLVA typing

Background  

Coxiella burnetii, the causative agent of Q fever, has a wide host range. Few epidemiological tools are available, and they are often expensive or not easily standardized across laboratories. In this work, C. burnetii isolates from livestock and ticks were typed using infrequent restriction site-PCR (IRS-PCR) and multiple loci variable number of tandem repeats (VNTR) analysis (MLVA).     

Synergy between type 1 fimbriae expression and C3 opsonisation increases internalisation of E. coli by human tubular epithelial cells

Background  

Bacterial infection of the urinary tract is a common clinical problem with E. coli being the most common urinary pathogen. Bacterial uptake into epithelial cells is increasingly recognised as an important feature of infection. Bacterial virulence factors, especially fimbrial adhesins, have been conclusively shown to promote host cell invasion. Our recent study reported that C3 opsonisation markedly increases the ability of E. coli strain J96 to internalise into human proximal tubular epithelial cells via CD46, a complement regulatory protein expressed on host cell membrane. In this study, we further assessed whether C3-dependent internalisation by human tubular epithelial cells is a general feature of uropathogenic E. coli and investigated features of the bacterial phenotype that may account for any heterogeneity.     

Highly sensitive real-time PCR for specific detection and quantification of Coxiella burnetii

Background  

Coxiella burnetii, the bacterium causing Q fever, is an obligate intracellular biosafety level 3 agent. Detection and quantification of these bacteria with conventional methods is time consuming and dangerous. During the last years, several PCR based diagnostic assays were developed to detect C. burnetii DNA in cell cultures and clinical samples. We developed and evaluated TaqMan-based real-time PCR assays that targeted the singular icd (isocitrate dehydrogenase) gene and the transposase of the IS1111a element present in multiple copies in the C. burnetii genome.     

Mass spectrometry-based quantitative proteomic analysis of <Emphasis Type="Italic">Salmonella enterica</Emphasis> serovar Enteritidis protein expression upon exposure to hydrogen peroxide

Background  

Salmonella enterica, a common food-borne bacterial pathogen, is believed to change its protein expression profile in the presence of different environmental stress such as that caused by the exposure to hydrogen peroxide (H₂O₂), which can be generated by phagocytes during infection and represents an important antibacterial mechanism of host cells. Among Salmonella proteins, the effectors of Salmonella pathogenicity island 1 and 2 (SPI-1 and SPI-2) are of particular interest since they are expressed during host infection in vivo and are important for invasion of epithelial cells and for replication in organs during systemic infection, respectively. However, the expression profiles of these proteins upon exposure to H₂O₂ or to host cells in vivo during the established phase of systemic infection have not been extensively studied.     

Nisin inducible production of listeriolysin O in <Emphasis Type="Italic">Lactococcus lactis</Emphasis> NZ9000

Background  

Listeria monocytogenesis a well-characterized food-borne pathogen that infects pregnant women and immunocompromised individuals. Listeriolysin O (LLO) is the major virulence factor of the pathogen and is often used as a diagnostic marker for detection of L. monocytogenes. In addition, LLO represents a potent antigen driving T cell-mediated immunity during infection. In the present work, Lactococcus lactisNZ9000 was used as an expression host to hyper-produce LLO under inducible conditions using the NICE (NIsin Controlled Expression) system. We created a modified pNZ8048 vector encoding a six-His-tagged LLO downstream of the strong inducible PnisA promoter.     

Killing of Mycobacterium avium subspecies paratuberculosis within macrophages

Background  

Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is a facultative intracellular pathogen that resides within host macrophages during infection of ruminant animals. We examined survival of M. paratuberculosis infections within cultured macrophages to better understand the interplay between bacterium and host.     

Phospholipase D promotes <Emphasis Type="Italic">Arcanobacterium haemolyticum</Emphasis> adhesion via lipid raft remodeling and host cell death following bacterial invasion

Background  

Arcanobacterium haemolyticum is an emerging bacterial pathogen, causing pharyngitis and more invasive infections. This organism expresses an unusual phospholipase D (PLD), which we propose promotes bacterial pathogenesis through its action on host cell membranes. The pld gene is found on a genomic region of reduced %G + C, suggesting recent horizontal acquisition.     

Epidemiology of Coxiella burnetii Infection in Africa: A OneHealth Systematic Review

Background

Q fever is a common cause of febrile illness and community-acquired pneumonia in resource-limited settings. Coxiella burnetii, the causative pathogen, is transmitted among varied host species, but the epidemiology of the organism in Africa is poorly understood. We conducted a systematic review of C. burnetii epidemiology in Africa from a “One Health” perspective to synthesize the published data and identify knowledge gaps.

Methods/Principal Findings

We searched nine databases to identify articles relevant to four key aspects of C. burnetii epidemiology in human and animal populations in Africa: infection prevalence; disease incidence; transmission risk factors; and infection control efforts. We identified 929 unique articles, 100 of which remained after full-text review. Of these, 41 articles describing 51 studies qualified for data extraction. Animal seroprevalence studies revealed infection by C. burnetii (≤13%) among cattle except for studies in Western and Middle Africa (18–55%). Small ruminant seroprevalence ranged from 11–33%. Human seroprevalence was <8% with the exception of studies among children and in Egypt (10–32%). Close contact with camels and rural residence were associated with increased seropositivity among humans. C. burnetii infection has been associated with livestock abortion. In human cohort studies, Q fever accounted for 2–9% of febrile illness hospitalizations and 1–3% of infective endocarditis cases. We found no studies of disease incidence estimates or disease control efforts.

Conclusions/Significance

C. burnetii infection is detected in humans and in a wide range of animal species across Africa, but seroprevalence varies widely by species and location. Risk factors underlying this variability are poorly understood as is the role of C. burnetii in livestock abortion. Q fever consistently accounts for a notable proportion of undifferentiated human febrile illness and infective endocarditis in cohort studies, but incidence estimates are lacking. C. burnetii presents a real yet underappreciated threat to human and animal health throughout Africa.     

The Coxiella burnetii type IV secretion system substrate CaeB inhibits intrinsic apoptosis at the mitochondrial level

Manipulation of host cell apoptosis is a virulence property shared by many intracellular pathogens to ensure productive replication. For the obligate intracellular pathogen Coxiella burnetii anti‐apoptotic activity, which depends on a functional type IV secretion system (T4SS), has been demonstrated. Accordingly, the C. burnetii T4SS effector protein AnkG was identified to inhibit pathogen‐induced apoptosis, possibly by binding to the host cell mitochondrial protein p32 (gC1qR). However, it was unknown whether AnkG alone is sufficient for apoptosis inhibition or if additional effector proteins are required. Here, we identified two T4SS effector proteins CaeA and CaeB (C . burnetii a nti‐apoptotic e ffector) that inhibit the intrinsic apoptotic pathway. CaeB blocks apoptosis very efficiently, while the anti‐apoptotic activity of CaeA is weaker. Our data suggest that CaeB inhibits apoptosis at the mitochondrial level, but does not bind to p32. Taken together, our results demonstrate that C. burnetii harbours several anti‐apoptotic effector proteins and suggest that these effector proteins use different mechanism(s) to inhibit apoptosis.     

A solute-binding protein for iron transport in <Emphasis Type="Italic">Streptococcus iniae</Emphasis>

Background  

Streptococcus iniae (S. iniae) is a major pathogen that causes considerable morbidity and mortality in cultured fish worldwide. The pathogen's ability to adapt to the host affects the extent of infection, hence understanding the mechanisms by which S. iniae overcomes physiological stresses during infection will help to identify potential virulence determinants of streptococcal infection. Grow S. iniae under iron-restricted conditions is one approach for identifying host-specific protein expression. Iron plays an important role in many biological processes but it has low solubility under physiological condition. Many microorganisms have been shown to be able to circumvent this nutritional limitation by forming direct contacts with iron-containing proteins through ATP-binding cassette (ABC) transporters. The ABC transporter superfamilies constitute many different systems that are widespread among living organisms with different functions, such as ligands translocation, mRNA translation, and DNA repair.     

Systems approach to investigating host-pathogen interactions in infections with the biothreat agent <Emphasis Type="Italic">Francisella</Emphasis>. Constraints-based model of <Emphasis Type="Italic">Francisella tularensis</Emphasis>

Background  

Francisella tularensis is a prototypic example of a pathogen for which few experimental datasets exist, but for which copious high-throughout data are becoming available because of its re-emerging significance as biothreat agent. The virulence of Francisella tularensis depends on its growth capabilities within a defined environmental niche of the host cell.     

The <Emphasis Type="Italic">Vibrio parahaemolyticus</Emphasis> Type III Secretion Systems manipulate host cell MAPK for critical steps in pathogenesis

Background  

Vibrio parahaemolyticus is a food-borne pathogen causing inflammation of the gastrointestinal epithelium. Pathogenic strains of this bacterium possess two Type III Secretion Systems (TTSS) that deliver effector proteins into host cells. In order to better understand human host cell responses to V. parahaemolyticus, the modulation of Mitogen Activated Protein Kinase (MAPK) activation in epithelial cells by an O3:K6 clinical isolate, RIMD2210633, was investigated. The importance of MAPK activation for the ability of the bacterium to be cytotoxic and to induce secretion of Interleukin-8 (IL-8) was determined.     

Nuclear trafficking of the anti‐apoptotic Coxiella burnetii effector protein AnkG requires binding to p32 and Importin‐α1

The obligate intracellular bacterium Coxiella burnetii causes the zoonotic disease Q‐fever. Coxiella pathogenesis depends on a functional type IV secretion system (T4SS). The T4SS effector AnkG inhibits pathogen‐induced host cell apoptosis, which is believed to be important for the establishment of a persistent infection. However, the mode of action of AnkG is not fully understood. We have previously demonstrated that binding of AnkG to p32 is crucial for migration of AnkG into the nucleus and that nuclear localization of AnkG is essential for its anti‐apoptotic activity. Here, we compared the activity of AnkG from the C. burnetii strains Nine Mile and Dugway. Although there is only a single amino acid exchange at residue 11, we observed a difference in anti‐apoptotic activity and nuclear migration. Mutation of amino acid 11 to glutamic acid, threonine or valine results in AnkG mutants that had lost the anti‐apoptotic activity and the ability to migrate into the nucleus. We identified Importin‐α1 to bind to AnkG, but not to the mutants and concluded that binding of AnkG to p32 and Importin‐α1 is essential for migration into the nucleus. Also during Coxiella infection binding of AnkG to p32 and Importin‐α1 is crucial for nuclear localization of AnkG.