Coxiella infection. In the following pages, I will

 

Coxiella burnetii
is a gram-negative intracellular
bacterium and the only member of the genus Coxiella.
Human disease caused by Coxiella is
termed ‘Q Fever’ or Query Fever 1. Since first described and isolated in the early
twentieth century, it has been characterized as a zoonotic agent capable of
infecting a diverse range of animals, from arthropods to mammals. Though it can
be transmitted via tick vector, most human infections result from inhalation of
aerosolized bacteria from farmed animals, especially cattle and sheep. Human
infection is usually asymptomatic or a mild self-limiting febrile illness.
However, those with heart valve defects, are immunocompromised, or pregnant may
experience a more severe acute illness after exposure and are at a higher risk
of persistent infection. In the following pages, I will describe the
epidemiology and molecular basis of infection with Coxiella burnetii. I will demonstrate the unique strategies the bacterium
employs to evade immune response to sustain persistent infection, as well as to
highlight the many virulence factors for initial colonization of the host.

 

I.    
History, Epidemiology, and Virulence Factor
Acquisition

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History

The first reported outbreak of ‘Q Fever’ occurred in
Australian slaughterhouse workers beginning in 1935. The workers presented with
a previously uncharacterized febrile illness. However, attempts to isolate the
causative agent failed; it was therefore presumed to be a viral agent. At the
same time, researchers at the Rocky Mountain Laboratory discovered a tick-borne
pathogen they called the ‘Nine-mile Agent,’ so named for the geographic area
the infected ticks were isolated. The researchers were unable to grow the agent
in pure culture, but they did determine that it was filterable, suggesting a
bacterial etiology. They developed a way to successfully passage the bacteria
in eggs. The two stories converged when a researcher at Rocky Mountain
developed a febrile illness from working with the Nine-mile Agent. They were
able to inoculate and replicate disease in guinea pigs with blood isolated from
the infected researcher, confirming that the Nine-mile Agent was the cause of
the researcher’s illness. Finally, after communicating with those studying the
Australian outbreak, they used tissue samples from Australia to challenge
guinea pigs exposed to the Nine-mile agent. The guinea pigs who were previously
exposed to the Nine-mile agent did not show symptoms of illness after exposure
to the Australian agent, confirming that the two illnesses were caused by the
same agent. Giemsa staining of infected animal tissue revealed a small,
rod-shaped intracellular bacterium. With the visualization complete, Koch’s
postulates were fulfilled, and the bacterium was confirmed as the cause of Q
Fever. The bacterium was named Coxiella
burnetii, after two of the researchers who first identified it: Dr. Herald
Cox, and Dr. Macfarlane Burnet. 1

 

Epidemiology

Coxiella
burnetii is a zoonotic pathogen with the capability to infect
birds, arthropods, and mammals. Most animals do not develop symptoms of
infection but remain long-term carriers of Coxiella.
Domesticated animals, including both farmed animals as well as cats and dogs,
are the most clinically relevant reservoirs for human transmission 2. Coxiella is distributed throughout the
world, apart from New Zealand. Human infection is most likely to occur due to
inhalation of aerosolized bacteria from infected animals. This is most likely
to occur in farming communities and rural areas. Most countries do not include
Coxiella burnetii infection as a
reportable disease. Because of the lack of required reporting, in addition to
the non-specific symptoms, Coxiella is likely more common than reported. The
bacterium is highly resistant to environmental exposure and has been shown to
be viable up to six weeks after exiting the host. Other, less common modes of
transmission include ingestion of contaminated raw milk, exposure to infected
wild animals, cats, and purported cases of blood-borne exposure.  Over forty genera of ticks are known to carry
Coxiella, but few if any cases of
tick-borne human transmission have been reported. Instead, it is hypothesized
that the tick is an important vector for the transmission between wild and
domesticated animals. There is little seasonal variation in the rates of Coxiella infection, though there is a
correlation between the seasonal birth rates of farmed animals and human
outbreaks due to the high bacterial load of placental tissue and the exposure
of humans to aerosolized bacteria during the birthing of infected animals.

 

Disease
Course and Treatment

Coxiella burnetii
is notable for an extremely low infectious dose, with an ID50 of fewer than 10 organisms 3. The
bacterium is thought to infect primarily macrophages and monocytes once
inhaled, and spread systematically with the circulation of the monocytes. Kupffer
cells of the liver have also been a reported reservoir of infection in the host
1. The
majority of exposed individuals are asymptomatic. Those that do have an average
incubation time of 20 days before symptoms develop. Symptoms are non-specific
and variable, but often include high fevers, fatigue, headache, and muscle
aches. Rarely, pneumonia, hepatitis acute endocarditis, or meningitis can
occur. Interestingly, pregnant women are more likely to be asymptomatic, but
there are an increased risk spontaneous
abortion or birth defects 4. In less
than five percent of patients, persistent infection occurs in the form of
endocarditis or bone/joint infection. Individuals with heart valve defects are
pregnant or immunocompromised are more likely to progress to chronic infection.
Treatment for most infected individuals is doxycycline, with trimethoprim-sulfamethoxazole used in pregnant
women due to the risk of birth defects with doxycycline. Alternatives include
macrolides or fluoroquinolones in
patients who are unable to tolerate doxycycline 5.

 

Genome,
Phylogenetics, and Virulence Acquisition

Coxiella was first
categorized as a Rickettsial organism due to its similarity to the lifecycle
and morphology of Rickettsia sp. Recent genetic analysis revealed that Coxiella is more closely related to Legionella pneumophila
and was reassigned to the Gammaproteobacteria
group 6. Within Coxiella, sixteen strains have been
characterized by restriction fragment length polymorphism (RFLP) analysis 1. All Coxiella strains contain a single
chromosome ranging from 1.5 to 2.4 Mb. All strains also have either a large
plasmid of 36 to 42 kb or have genetic elements from the plasmid integrated
into the genome 1. There is
limited genomic heterogeneity between strains, though
a comparative analysis of 4 different isolates by Beare et. al. show that
isolates vary greatly by their repertoire of pseudogenes 7. The same
analysis revealed the relative lack of conjugal apparatus and the presence of
genes that confer natural competence (comA).
They posit that the high number of insertional sequences and pseudogenes are
evidence of transposon rearrangement as the main driver of genetic diversity.
Phage transduction has not been observed.

 

 

 

 

II.  
Host Defense & Pathogen Evasion

 

Host Defense Mechanisms

Coxiella
primarily targets and infects macrophages and monocytes but can also infect
dendritic cells. It has many mechanisms to reduce the inflammatory response in
these innate immune populations, including evasion of phagolysosomal killing.
Therefore, host must rely on adaptive immunity to eliminate the infection, similar
to other intracellular agents. This is demonstrated by the finding that SCID
mice are unable to control Coxiella
infection. SCID mice lack T and B cells and cannot mount an adaptive response. In
humans, immunocompromised status is associated with the persistence of
infection, consistent with the study of SCID mice. The adaptive immune response
is primarily cell-mediated. Both CD4+ and CD8+ T cells are involved in mounting
a response. Interferon gamma (IFNg) is the primary cytokine associated with a
successful immune response and has been shown to be required in the mouse model
of infection 8. The exact role of IFNg in immunity isn’t entirely
clear; IFNg is thought to mediate the clearance of infection by improving the
efficiency of macrophage phagosome-lysosome fusion and the restoration of
lysosomal pH, both of which are partially blocked by Coxiella. However, in a study by Cunha et. al., IFNg caused reduced
replication of bacteria when administered to the lung and a dampened immune
response with significant disease-associated weight-loss if given systemically 9. CD4+ T cells are polarized to the Th1 response
during infection, which is consistent with the importance of IFNg, the primary
effector cytokine of Th1s. Tumor necrosis factor (TNF) is also produced by the
adaptive immune response, which induces apoptosis in infected macrophages. Cell-mediated
immunity (largely dependent on cytotoxic T cells) has been shown to be critical
to prevent the progression of chronic
infection.  Interestingly, the antibody
response is not required for clearance of the bacteria and, in the case of
persistent infection, may dampen the immune response by producing IL-10, an
anti-inflammatory cytokine 8.

 

Pathogen Evasion

Coxiella
is first able to evade host immunity by infecting intracellularly. This reduces
the efficiency of immune surveillance by antigen presenting cells and delays
the recruitment of the adaptive immune response, which is required for
clearance. Intracellular infection of mobile macrophages and monocytes also
allow the infection to spread systemically without detection. In fact, bacteria
continue to be shed from individuals that have ‘cleared’ the infection,
possibly because of a remaining bone marrow derived macrophage reservoir 10. The bacterium targets and invades macrophages by
inducing phagocytosis. This usually occurs in the alveolar macrophages of the
lung 1. Once inside the host cell, the bacterium
circumvents bacterial killing by secreting effector proteins into the host
cytosol via a Dot/Icm-like T4SS. These effectors block apoptosis of the host
cell by blocking the activation of apoptotic mediators caspase-3, caspase-9,
and PARP. One such effector identified is IcaA, which inhibits caspase-11
dependent activation of the NLRP3 inflammasome in host cells 9. Other studies have demonstrated alterations in
NF-kB localization and kinase activation, though it is unknown how this
maintains the intracellular infection 10. Bacterial effectors homologous to other pathogens
have not been identified in Coxiella and
few pathogen-specific effectors have characterized.  Inside the vacuole, the contents are allowed to acidify to a
pH of 5 10. Proteolytic enzymes and hydrolases also accumulate.
Despite this seemingly inhospitable environment, Coxiella actively regulates and maintains these conditions as a
replication niche termed the parasitophorous vacuole (PV). Within the vacuole,
the pathogen switches metabolic states from a hearty small colony variant,
suitable for harsh abiotic surfaces, to a large, replicative form. The bacteria
divide rapidly inside the PV, which presents a new problem: limited space and
nutrients in the vacuole. The bacterium recruits a ‘nutrient delivery system’
to circumvent this problem. The pathogen recruits autophagosomes to fuse with
the PV, providing the lipid membrane needed to expand and nutrient-rich
contents 10.

 

Finally, Coxiella alters host cell behavior and
global host immunity to reduce the effectiveness of the host response. This can
lead to persistence and chronic infection. For instance, initial successful
invasion of Coxiella into monocytes
polarizes their differentiation to a ‘M2’ phenotype, resulting in less
efficient killing of the bacteria and production of immune-dampening IL-10 11. IL-10 is important for maintenance of the chronic
infection of Coxiella.

 

III.Secretion
Systems

 

Secretion Systems

Because of its fastidious
growth conditions, most studies of virulence factors have been based on a
genomic analysis 12. The study of Coxiella
secretion systems is no different. Coxiella
is predicted to contain functional T1SS, and T4SS, with components of the type
IV pilus apparatus.