Monitoring Laboratory Occupational Exposures to Burkholderia pseudomallei.

Background: Burkholderia pseudomallei is a Tier 1 overlap select agent and subject to the select agent regulations (42 CFR §73 and 9 CFR §121). It is a gram-negative, motile, soil saprophyte, and the etiologic agent of melioidosis. B. pseudomallei infection can produce systemic illness and can be fatal in the absence of appropriate treatment. Laboratory exposures involving this organism may occur when appropriate containment measures are not employed. Current disease treatment inadequacies and the risk factors associated with melioidosis make this an agent of primary concern in research, commercial, and clinical laboratory environments. Results: This study presents data reported to Centers for Disease Control and Prevention (CDC), Division of Select Agents and Toxins for releases involving B. pseudomallei in 2017-2019 that occurred in Biosafety Level (BSL)-2 and BSL-3 laboratories. Fifty-one Animal and Plant Health Inspection Service (APHIS)/CDC Form 3 release reports led to the medical surveillance of 275 individuals. Entities offered post-exposure prophylaxis to ∼76% of the individuals impacted in the presented events. Summary: Laboratory safety can be improved by implementing appropriate safety precautions to minimize exposures. Most of the incidents discussed in this evidence-based report occurred during work conducted in the absence of primary containment. None of the releases resulted in illness, death, or transmission to or among workers, nor was there transmission outside of a laboratory into the surrounding environment or community. Effective risk assessment and management strategies coupled with standard and special microbiological policies and procedures can result in reduced exposures and improved safety at facilities.

A Toxoplasma palmitoyl acyl transferase and the palmitoylated armadillo repeat protein TgARO govern apical rhoptry tethering and reveal a critical role for the rhoptries in host cell invasion but not egress.

Apicomplexans are obligate intracellular parasites that actively penetrate their host cells to create an intracellular niche for replication. Commitment to invasion is thought to be mediated by the rhoptries, specialized apical secretory organelles that inject a protein complex into the host cell to form a tight-junction for parasite entry. Little is known about the molecular factors that govern rhoptry biogenesis, their subcellular organization at the apical end of the parasite and subsequent release of this organelle during invasion. We have identified a Toxoplasma palmitoyl acyltransferase, TgDHHC7, which localizes to the rhoptries. Strikingly, conditional knockdown of TgDHHC7 results in dispersed rhoptries that fail to organize at the apical end of the parasite and are instead scattered throughout the cell. While the morphology and content of these rhoptries appears normal, failure to tether at the apex results in a complete block in host cell invasion. In contrast, attachment and egress are unaffected in the knockdown, demonstrating that the rhoptries are not required for these processes. We show that rhoptry targeting of TgDHHC7 requires a short, highly conserved C-terminal region while a large, divergent N-terminal domain is dispensable for both targeting and function. Additionally, a point mutant lacking a key residue predicted to be critical for enzyme activity fails to rescue apical rhoptry tethering, strongly suggesting that tethering of the organelle is dependent upon TgDHHC7 palmitoylation activity. We tie the importance of this activity to the palmitoylated Armadillo Repeats-Only (TgARO) rhoptry protein by showing that conditional knockdown of TgARO recapitulates the dispersed rhoptry phenotype of TgDHHC7 knockdown. The unexpected finding that apicomplexans have exploited protein palmitoylation for apical organelle tethering yields new insight into the biogenesis and function of rhoptries and may provide new avenues for therapeutic intervention against Toxoplasma and related apicomplexan parasites.

The moving junction protein RON8 facilitates firm attachment and host cell invasion in Toxoplasma gondii.

The apicomplexan moving junction (MJ) is a highly conserved structure formed during host cell entry that anchors the invading parasite to the host cell and serves as a molecular sieve of host membrane proteins that protects the parasitophorous vacuole from host lysosomal destruction. While recent work in Toxoplasma and Plasmodium has reinforced the composition of the MJ as an important association of rhoptry neck proteins (RONs) with micronemal AMA1, little is known of the precise role of RONs in the junction or how they are targeted to the neck subcompartment. We report the first functional analysis of a MJ/RON protein by disrupting RON8 in T. gondii. Parasites lacking RON8 are severely impaired in both attachment and invasion, indicating that RON8 enables the parasite to establish a firm clasp on the host cell and commit to invasion. The remaining junction components frequently drag in trails behind invading knockout parasites and illustrate a malformed complex without RON8. Complementation of Δron8 parasites restores invasion and reveals a processing event at the RON8 C-terminus. Replacement of an N-terminal region of RON8 with a mCherry reporter separates regions within RON8 that are necessary for rhoptry targeting and complex formation from those required for function during invasion. Finally, the invasion defects in Δron8 parasites seen in vitro translate to radically impaired virulence in infected mice, promoting a model in which RON8 has a crucial and unprecedented task in committing Toxoplasma to host cell entry.

Novel components of the Apicomplexan moving junction reveal conserved and coccidia-restricted elements.

Apicomplexan parasites generally invade their host cells by anchoring the parasite to the host membrane through a structure called the moving junction (MJ). This MJ is also believed to sieve host proteins from the nascent parasitophorous vacuole membrane, which likely protects the pathogen from lysosomal destruction. Previously identified constituents of the Toxoplasma MJ have orthologues in Plasmodium, indicating a conserved structure throughout the Apicomplexa. We report here two novel MJ proteins, RON5 and RON8. While RON5 is conserved in Plasmodium, RON8 appears restricted to the coccidia. RON8, which is likely essential, co-immunoprecipitates RON5 and known MJ proteins from extracellular parasites, indicating that a preformed complex exists within the parasites. Upon secretion, we show that RON8 within the MJ localizes to the cytoplasmic face of the host plasma membrane. To examine interactions between RON8 and the host cell, we expressed RON8 in mammalian cells and show that it targets to its site of action at the periphery in a manner dependent on the C-terminal portion of the protein. The discovery of RON5 and RON8 provides new insight into conserved and unique elements of the MJ, furthering our understanding of how the MJ contributes to the intricate mechanism of Apicomplexan invasion.