Vazquez, 19, made 13 appearances as a substitute as a forward/winger in 2017 and scored just a minute into his debut at Real Salt Lake on April 22. He added two goals an assist in two US Open Cup starts. 2017 (Atlanta United): Appeared in 13 games, registering one goal and one assist. Scored one minute into his MLS debut in a 3-1 win at Real Salt Lake on April 22. Assisted on Josef Martinez’s game-winning goal in 1-0 win against Colorado on June 24. US Open Cup: Tallied two goals and an assist in two starts. Started, scored the game-winning goal and added an assist in 90 minutes on June 14 against Charleston.
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Started and scored in 90 minutes against Miami FC on June 28. Born in Chula Vista, Calif., he made his U.S.
National Team debut on Feb. 20, 2015 with the U-17s during a 5-2 win over Bermuda. Two days later in a rematch, Vazquez scored a hat-trick in a 4-0 rout. Later that year, he was named to head coach Richie Williams’ 21-man roster for the 2015 FIFA U-17 World Cup in Chile.
Despite failing to reach the knockout rounds, Vazquez played a huge role for the U.S., scoring two of their three goals in group play. He gave the U.S. A 2-0 lead against Croatia on Oct. 20, 2015 and put the U.S. Ahead of Chile three days later.
The only other goal scored in the tournament by the U.S. Was by Borussia Dortmund midfielder Christian Pulisic. In 2016 with the U.S. U-19s, Vazquez scored four goals, including the game-winner against Spain in the 2016 COTIF Tournament, a highlight-reel free kick from 30 yards out to defeat the hosts in group play. This was Spain’s only loss in the tournament as they went on to defeat Argentina in the Final. Vazquez also scored in September against Serbia in the Stevan-Vilotic Cele Tournament.
8, 2016 Vazquez was called up to the U.S. U-20 National Team for the December international training camp in San Jose, Costa Rica.
Most intracellular parasites employ sophisticated mechanisms to direct biogenesis of a vacuolar replicative niche that circumvents default maturation through the endolysosomal cascade. However, this is not the case of the Q fever bacterium, Coxiella burnetii. This hardy, obligate intracellular pathogen has evolved to not only survive, but to thrive, in the harshest of intracellular compartments: the phagolysosome. Following internalization, the nascent Coxiella phagosome ultimately develops into a large and spacious parasitophorous vacuole (PV) that acquires lysosomal characteristics such as acidic pH, acid hydrolases and cationic peptides, defences designed to rid the host of intruders. However, transit of Coxiella to this environment is initially stalled, a process that is apparently modulated by interactions with the autophagic pathway. Coxiella actively participates in biogenesis of its PV by synthesizing proteins that mediate phagosome stalling, autophagic interactions, and development and maintenance of the mature vacuole.
Among the potential mechanisms mediating these processes is deployment of a type IV secretion system to deliver effector proteins to the host cytosol. Here we summarize our current understanding of the cellular events that occur during parasitism of host cells by Coxiella. Introduction The obligate intracellular bacterium Coxiella burnetii is the causative agent of the zoonosis Q fever, a disease that generally manifests as an acute, debilitating flu‐like illness ( ).
Unlike other obligate intracellular pathogens, Coxiella is highly resistant to environmental stresses such as high temperature, osmotic pressure and ultraviolet light (; ). These characteristics are attributed to a small cell variant (SCV) form of the organism that is part of a biphasic developmental cycle including a more metabolically and replicatively active large cell variant (LCV) form (;; ). Enhanced stability outside the host cell facilitates the primary mode of human infection which is inhalation of contaminated aerosols. The organism is highly infectious with an infectious dose approaching one organism ( ). Extracellular stability correlates with Coxiella replication in the most inhospitable compartment of the host cell – the phagolysosome. In fact, Coxiella is the only known example of a bacterial pathogen that replicates throughout its infectious cycle in a parasitophorous vacuole (PV) that is indistinguishable from a secondary lysosome (;; ). Lipopolysaccharide phase variation The ability of Coxiella to prosper in a normally bacteriostatic/cidal vacuole is central to its pathogenesis.
Kayla Vazquez
Unfortunately, our understanding of molecular mechanisms employed by Coxiella to persist in this environment is limited by the lack of a system to genetically manipulate the organism. Currently, lipopolysaccharide (LPS) is the only defined Coxiella virulence factor ( ). Virulent ‘phase I’ organisms isolated from natural sources and infections produce a full‐length LPS.
Serial in vitro passage of phase I Coxiella in embryonated eggs or tissue culture results in bacteria that produce LPS molecules of decreasing molecular weight, culminating in the severely truncated LPS of avirulent ‘phase II’ organisms ( ). A stable isogenic phase II LPS variant (Nine Mile, phase II, clone 4, RSA439) of the virulent Nine Mile phase I reference strain (RSA493) has been cloned that is exempt from US Centers for Disease Control and Prevention select agent regulations and is suited for work at biosafety level 2 ( ). All other Coxiella strains are considered biosafety level 3 organisms ( ). Coxiella RSA439 contains a chromosomal deletion that eliminates genes involved in O‐antigen biosynthesis, a defect that is associated with attenuated virulence (; ). However, the rough LPS chemotype and resultant avirulence of this strain is likely due to an additional point/frameshift mutation, small deletion or transposon insertion in a gene earlier in the LPS biosynthetic pathway (; ).
In addition to a lower bio‐containment level, RSA439 has the advantage over phase I organisms for in vitro cellular biology studies by being ∼10‐fold more infectious for cultured cells ( ). Moreover, with the exception of primary murine macrophages, most studies indicate that phase I and phase II organisms replicate with similar kinetics in phenotypically indistinguishable lysosome‐like PV (discussed below). The virulence properties of full‐length Coxiella LPS are related to the molecule's ability to shield the outer membrane.
For example, phase II, but not phase I, organisms are readily killed via the complement membrane attack complex ( ). Additionally, antibodies to Coxiella surface proteins are sterically inhibited from binding phase I organisms, an effect that is reversed if LPS is first chemically extracted ( ). Phase I LPS also masks Coxiella toll‐like receptor (TLR) ligands from innate immune recognition by dendritic cells ( ) and may also inhibit Coxiella interaction with the CR3 ( α M β 2 integrin) receptor of macrophages ( ). Phase I and phase II LPS lipid A moieties are chemically identical and the molecule not only fails to ligate TLR‐4 (; ), but is antagonistic against TLR‐4 signalling by other LPS ( ). Relative to phase I Coxiella, phase II organisms display growth defects in primary mouse macrophages (;;; ). This growth restriction appears to be murine‐specific as phase variants grow at similar rates in primary macrophages from guinea pigs ( ), non‐human primates and humans (J. Shannon et al., manuscripts in preparation).
Phase II Coxiella may specifically activate inhibitory innate immune mediators in primary mouse cells that limit growth ( ). Consistent with this idea is the observation that primary mouse macrophages treated with nitric oxide synthase inhibitors, or from TLR‐2 knockout mice, are markedly more permissive for growth of phase II Coxiella than untreated or wild‐type macrophages, respectively (; ). Moreover, phase II Coxiella grows moderately better in macrophages from A/J and BALB/c mice, which have known deficiencies in innate immunity ( ), than those from immunocompetent C57BL/6 mice ( ). In vitro and in vivo model systems In vitro, Coxiella promiscuously infects a wide variety of cell types, and many epithelial and fibroblast‐like continuous cell lines have been used as models to study Coxiella–host interactions. Cell culture systems supporting Coxiella growth include Vero (African green monkey kidney epithelial) ( ), BHK‐21 (hamster kidney fibroblast) ( ), L‐929 (murine fibroblast) ( ), HEL (human embryonic lung fibroblast) ( ), HeLa (human cervical epithelial) ( ) and CHO (Chinese hamster ovary fibroblast) ( ) cells. In natural infections, Coxiella has a tropism for cells of the mononuclear phagocyte system such as alveolar macrophages of the lung and Kupffer cells of the liver (; ), with organisms also infrequently observed in pneumocytes, fibroblasts and endothelial cells ( ). Consequently, monocyte/macrophage‐like cell lines including J774A.1 (murine macrophage‐like) ( ), P388D1 (murine macrophage‐like) ( ) and THP‐1 (human monocyte‐like) ( ) cells have been extensively employed to more accurately mimic the in vivo situation, with more recent studies focused on interactions with primary human monocytes/macrophages ( ) and dendritic cells ( ).
With a few notable exceptions (discussed below), the findings of these studies concur and have cumulatively allowed modelling of the Coxiella infectious cycle. The most accurate rodent model of human Q fever is the guinea pig. Inoculation with as few as 10 phase I Coxiella organisms results in a symptomatic self‐limiting infection that closely mimics clinical human acute Q fever with signs including sustained elevated fever (;; ). However, a limitation of the guinea pig model is the lack of genetic and immunological tools that are the mainstay of mouse models of infectious disease. As determined by sero‐conversion, inbred mouse strains are readily infected by low doses of Coxiella ( ). However, higher doses ( 1 × 10 5 organisms) are required before strains that are both resistant (e.g. C57BL/6) and sensitive (e.g.
A/J) to lethal Coxiella infection show clinical signs of infection such as pronounced splenomegaly. Nonetheless, splenomegaly is a reliable readout of the extent of Coxiella replication (; ) and, among other applications, this system has been employed to test Q fever subunit vaccine candidates ( ). Moreover, in conjunction with infection of knockout, athymic and SCID mice, splenomegaly and other indicators of infection such as granuloma formation have been assessed to define important components of the innate and adaptive immune response to Coxiella (;;;; ) and the pathogenic potential of different Coxiella isolates ( ). Pulmonary infection of cynomolgus macaques is the established non‐human primate model of Q fever ( ); however, expense and biosafety level 3 animal care and use issues severely restrict its use. Coxiella adherence and internalization Coxiella internalization into host cells occurs by microfilament‐dependent endocytosis (;; ).
Adherence of virulent phase I Coxiella to THP‐1 human monocyte‐like cells results in dramatic reorganization of the actin cytoskeleton that induces pronounced membrane protrusions at the site of bacterial attachment ( ). Similar cellular effects are not observed upon adherence of avirulent phase II organisms ( ). Adherence of phase I Coxiella also activates host protein tyrosine kinases with the resulting phosphorylated host proteins localizing to filamentous actin within the protrusions ( ). Inhibition of protein tyrosine kinase activity reduces membrane protrusion formation and enhances phase I phagocytosis ( ).
Pathogen‐induced actin reorganization resulting in membrane protrusions, such as the membrane ruffling associated with Salmonella spp. Infection ( ), is generally considered a mechanism that increases the efficiency of pathogen uptake.
However, membrane protrusion‐inducing phase I Coxiella are internalized less efficiently than phase II organisms by both professional phagocytes ( ) and fibroblast/epithelial cells (; ). In professional phagocytes, this discrepancy may reflect differential engagement of host cell receptors by phase variants. The THP‐1 receptor for phase I organisms is the leukocyte response integrin α v β 3 whereas phase II organisms additionally engage the CR3 receptor ( ). Membrane projections induced by phase I adherence are proposed to restrict engagement of the CR3 coreceptor, thereby lowering the efficiency of internalization ( ). Full‐length LPS of phase I organisms may further restrict binding of CR3 by a critical Coxiella ligand as the molecule is known to sterically mask Coxiella surface proteins (; ). Adherence and internalization of phase II organisms by non‐phagocytic cells, such as Vero epithelial cells and L‐929 fibroblasts, also occurs at much higher rates than phase I organisms. However, differential engagement of integrin α v β 3 and CR3 cannot account for increased phase II uptake as these cells lack these receptors.
Phase II Coxiella produce a truncated LPS with a much lower carbohydrate content than phase I organisms, rendering phase II highly hydrophobic ( ). Consequently, increased phagocytosis of phase II Coxiella by all host cell types is likely augmented by non‐specific hydrophobic interactions that facilitate interactions with the host plasma membrane and cognate receptors ( ). The Coxiella ligand(s) mediating uptake is unknown but is likely proteinaceous as pretreatment of the organism with proteases dramatically inhibits internalization ( ). Coxiella phagosome maturation Following internalization, the nascent Coxiella‐containing phagosome transits through the default endocytic cascade to ultimately fuse with the lysosomal compartment ( ). Sequential recruitment by the Coxiella phagosome is observed for the small GTPases Rab5 and Rab7, which are prototypic markers of early and late endosomes, respectively, that regulate membrane trafficking ( ).
However, fusion with lysosomes, as indicated by the presence of the lysosomal enzymes acid phosphatase ( ) and cathepsin D ( ), takes approximately 2 h, which is significantly slower than phagosomes harbouring inert particles such as latex beads that acquire lysosomal enzymes in about 15 min ( ). Exciting new studies from the laboratory of M. Colombo suggest that delayed lysosome interactions are due to early engagement between the Coxiella phagosome and the autophagic pathway (;; ). Indeed, the Coxiella phagosomal membrane is decorated with the autophagosome marker microtubule‐associated protein light‐chain 3 (LC3) as early as 5 min post infection (; ).
Early autophagosome interactions and delayed lysosomal hydrolase delivery are reliant on Coxiella protein synthesis (; ), indicating these are pathogen‐directed processes. Autophagy is normally employed to remove defective organelles and cytoplasmic material via trafficking of this material to autophagolysosomes ( ), and pathogen interactions with autophagosomes can have either beneficial or detrimental effects (; ). Coxiella clearly benefits from autophagy as induction of this pathway increases the number of infected cells, the size of the PV, and the extent of Coxiella replication ( ). These effects are abolished when infected cells are treated with the autophagy inhibitors wortmannin and 3‐methyladenine ( ). The advantage to Coxiella of autophagy‐mediated delay of phagosome maturation is unclear as the organism prospers in the normally toxic lysosomal environment. Phagosome stalling has been speculated to allow morphological differentiation of the resistant, metabolically sluggish SCV form of Coxiella to the metabolically and replicatively active LCV developmental form (; ).
A similar ‘pregnant pause’ in phagosome maturation is associated with the vacuoles of Leishmania spp. And Legionella and is speculated to provide these organisms adequate time to differentiate into replicative forms that thrive in lysosomes ( ). However, differentiation of SCV to LCV takes 1–2 days to occur ( ), which is considerably longer than the few hours of phagosome stalling.
Kayla Vazquez Kissimmee
Other possible benefits of Coxiella interactions with the autophagic pathway include delivery of nutrients and membrane. Because the Coxiella PV is not passively permeable to small molecules ( ), early interactions with autophagosomes may deliver a critical pulse of peptides/amino acids and saccharides that upregulate Coxiella metabolism and initiate SCV to LCV differentiation. Later interactions likely provide sustained metabolites for LCV exponential replication (; ).
Moreover, Coxiella does not synthesize its own PV membrane; thus, multiple fusion events with autophagosomes along with endolysosomal vacuoles are likely essential to provide sufficient membrane for the enlarging PV. Type IV secretion As mentioned earlier, Coxiella protein synthesis is required for early autophagosome interactions that promote stalling of PV maturation. Protein effectors of this process presumably target host factors that regulate vesicular trafficking and are likely delivered to the cytosol via the activity of a type IV secretion system (T4SS) that has homology to the Dot/Icm T4SS of Legionella ( ).
Dot/Icm T4SS function is essential for establishment of the replication vacuole of Legionella (; ), and T4SSs are employed by a number of Gram‐negative pathogens to translocate proteins that modulate specific host processes for the pathogen's benefit ( ). Conservation of the Dot/Icm T4SS between Coxiella and Legionella is consistent with the recently established close phylogenetic relatedness of the two organisms ( ). The Coxiella genome contains 23 of the 26 Legionella dot/ icm genes with the exception of icmR, dotJ and dotV ( ). Coxiella dotB, icmS, icmW and icmT complement corresponding mutants in Legionella. However, Coxiella icmX, icmQ, dotM, dotL, dotN and dotO do not complement, suggesting there are interactions between functional Coxiella Dot/Icm T4SS components that are not conserved in Legionella (; ). The lack of complementation by Coxiella icmQ was initially attributed to failure of the encoded protein to interact with IcmR, a chaperone of Legionella IcmQ that is absent in Coxiella ( ). However, a recent study shows that both Coxiella and Legionella produce proteins that are non‐homologous, but functionally similar, to IcmR ( ).
To date, no secretion substrates have been described for the Coxiella Dot/Icm T4SS. Moreover, Coxiella does not contain homologues of Legionella type IV substrates including RalF ( ) and SidM/DrrA ( ) (guanine exchange factors), VipD, VpdA and VpdB (phospholipases) ( ), and the paralogue Sde (SdeA–C) ( ) and Sid (SidA‐H) ( ) protein families.
A consistent theme among secreted bacterial effector molecules is the presence of eukaryotic‐like motifs that functionally mimic the activity of host cell proteins ( ). Exploiting this property, a bioinformatic screen recently identified new Legionella T4SS substrates that were likely acquired by interdomain horizontal gene transfer ( ). Novel effector molecules include protein families possessing coiled‐coil domains (CCD), tetratricopeptide repeats (TPR), leucine‐rich repeats and ankyrin repeats (Anks). A common property of these protein motifs is their involvement in multiple types of protein–protein interactions and signalling pathways. A similar in silico analysis reveals that the sequenced Nine Mile I Coxiella genome also encodes a large number of proteins containing eukaryotic‐like motifs that may be potential T4SS effectors.
These include at least eight TPR proteins (e.g. CBU1364) and 10 CCD proteins (e.g. Moreover, 20 Anks were revealed (e.g. CBU0781), which is seven more than originally highlighted in the Coxiella genome study ( ).
These candidate T4SS effector molecules await testing in heterologous systems and/or gene knockout experiments upon development of Coxiella genetics. Legionella expression of the Dot/Icm T4SS coincident with, and immediately following, infection of macrophages is critical for biogenesis of a PV that supports growth (; ). In vivo, Coxiella is metabolically quiescent outside of a moderately acidic host cell vacuole ( ); thus, it is unclear how type IV secretion, an energy‐dependent process ( ), could be involved in cellular uptake. However, secretion of a preloaded type III effector molecule by metabolically inert elementary bodies of Chlamydia trachomatis that induces their uptake has recently been reported ( ), and a similar event may occur during Coxiella internalization.
Early in the infectious process, Coxiella protein synthesis is clearly required for early autophagosome interactions, phagosome stalling (; ), and homotypic fusion of individual Coxiella phagosomes in multiply infected cells ( ). Thus, the organism must be metabolically activated early during transit through the endocytic pathway, with the potential to synthesize de novo T4SS effectors that modulate these processes. Coxiella Dot/Icm T4SS may also be required late in the pathogen's infectious cycle as the PV collapses and fuses less efficiently with latex bead‐containing phagosomes in the absence of Coxiella protein synthesis ( ). Consistent with this idea is the observation that dotA is expressed during Coxiella's stationary growth phase ( ). A late role for Legionella Dot/Icm secretion in delaying macrophage apoptosis has recently been defined (; ), with one effector (SdhA) identified that appears to act in part by suppressing caspase activity ( ).
Anti‐apoptotic effects may also be mediated by the Coxiella Dot/Icm T4SS as persistently infected macrophages show little cytopathic effect ( ). Features of the mature Coxiella PV Based on cytochemical localization of the lysosomal enzymes acid phosphatase and 5′‐nucleotidase in infected mouse L‐929 cells, Burton and coworkers ( ) were the first to suggest Coxiella replicates in a phagolysosome. This hypothesis was later buttressed by the results of Akporiaye et al. ( ) who demonstrated in infected J774A.1 mouse macrophages that the Coxiella vacuole acidifies and acquires thorium dioxide from secondary lysosomes. The membrane of the mature PV ( 2 days post infection) has been subsequently shown to decorate with the vacuolar type H + ATPase ( ), Rab7 (; ), three lysosomal glycoproteins LAMP‐1 and LAMP‐2 and LAMP‐3 (CD63) (;;; ), flotillin 1 and 2 ( ), and the autophagosome markers LC3 ( ) and Rab24 ( ).
Differential trafficking has been proposed for Coxiella phase variants in resting primary human monocytes and THP‐1 human monocyte‐like cells ( ). Based on the presence of active cathepsin D, this study suggests maturation of phagosomes containing phase I Coxiella is impaired and stops at a growth‐permissive late endosomal stage. Conversely, phagosomes sheltering phase II Coxiella are thought to fully mature to a microbicidal lysosomal compartment, a process presumably directed by the strain's selective engagement and activation of the CR3 receptor (; ). The results of other studies conflict with this proposal. First, phase II variants grow robustly in a number of CR3‐expressing murine macrophage‐like cells ( ). Second, phase II Coxiella replicate in cathepsin D‐positive vacuoles in CHO cells ( ). Third, vacuoles harbouring replicating phase I bacteria in murine L‐929 and J774A.1 cells contain lysosomal acid hydrolases, indicating full endolysosomal maturation (;; ).
Finally, we find that phase I and phase II Coxiella grow with similar kinetics in human monocyte‐derived dendritic cells ( ) and macrophages where they traffic to, and replicate within, the same vacuole (J. Shannon et al., manuscript in preparation). Species‐specific trafficking behaviour and/or differences in primary macrophage isolation and cultivation may account for these disparate findings.
Nonetheless, differential trafficking as a mechanism that contributes to the virulence properties of phase variants is an intriguing possibility that deserves further investigation. The volume of the mature Coxiella PV is striking. The vacuole enlarges to occupy nearly the entire cytoplasm with a volume usually well in excess of the resident organisms. During division of an infected cell, the PV segregates to one daughter cell, leaving the companion daughter cell parasite‐free ( ). This appears to be the case even for the Priscilla strain of Coxiella which resides in an unusual multilobed vacuole ( ). Among other possibilities, generation of spacious PV by intracellular parasites is proposed to dilute toxic lysosomal compounds, thereby rendering them less effective ( ). Transmission electron micrographs of phase II Coxiella‐infected Vero cells.
Cells were infected for 2 days (A) or 6 days (B–D), then examined via transmission electron microscopy. Panel A shows a spacious PV at 2 days post infection (dpi) harbouring a few Coxiella organisms (arrow). Multilamellar bodies (arrowhead) are also present that may result from PV fusion with autophagosomes. By 6 dpi (B and C), the PV has enlarged to occupy a large portion of the host cell and contains numerous Coxiella that have entered the stationary phase of their growth cycle ( ). Panels C and D (inset) demonstrate the close association between individual Coxiella and the PV membrane that may facilitate secretion of type IV effector proteins into the host cell cytosol. The remarkable redistribution of host lipid components to the PV in the absence of obvious cytopathic effects and perturbation of the host cell cycle (; ) is an exquisite example of parasite‐host coevolution.
However, relative to protein constituents, the source and composition of lipids comprising Coxiella and other parasite PV are poorly defined. A recent study addressed this gap in knowledge by examining cholesterol metabolism in Coxiella‐infected Vero cells ( ).
Filipin staining indicates the PV membrane has approximately the same cholesterol content as the cholesterol‐rich plasma membrane ( ). To compensate for biogenesis of the novel Coxiella compartment, infected cells produce 73% more cholesterol late in the pathogen's infectious cycle (6 days post infection) than uninfected cells, indicating a dramatic increase in cholesterol metabolism as opposed to simple redistribution of normal cholesterol stores. Accordingly, transcription of host genes involved in both cholesterol uptake (e.g. LDL receptor) and biosynthesis (e.g.
Lanosterol synthase) increases in response to the growth cycle of Coxiella, with upregulation of these genes observed only during log phase (∼4 days post infection), a time when the PV is dramatically expanding (.