Sad1 is an essential splicing factor initially identified in a genetic screen in Saccharomyces cerevisiae for snRNP assembly defects. Based on sequence homology, Sad1, or USP39 in humans, is predicted to comprise two domains: a zinc finger ubiquitin binding domain (ZnF-UBP) and an inactive ubiquitin-specific protease (iUSP) domain, both of which are well conserved. The role of these domains in splicing and their interaction with ubiquitin are unknown. We first used splicing microarrays to analyze Sad1 function in vivo and found that Sad1 is critical for the splicing of nearly all yeast intron-containing genes. By using in vitro assays, we then showed that it is required for the assembly of the active spliceosome. To gain structural insights into Sad1 function, we determined the crystal structure of the full-length protein at 1.8 Å resolution. In the structure, the iUSP domain forms the characteristic ubiquitin binding pocket, though with an amino acid substitution in the active site that results in complete inactivation of the enzymatic activity of the domain. The ZnF-UBP domain of Sad1 shares high structural similarly to other ZnF-UBPs; however, Sad1's ZnF-UBP does not possess the canonical ubiquitin binding motif. Given the precedents for ZnF-UBP domains to function as activators for their neighboring USP domains, we propose that Sad1's ZnF-UBP acts in a ubiquitin-independent capacity to recruit and/or activate Sad1's iUSP domain to interact with the spliceosome.

In this study, we developed angiotensin-converting enzyme 2 (ACE2)-specific, peptide-derived ⁶⁸Ga-labeled radiotracers, motivated by the hypotheses that ACE2 is an important determinant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) susceptibility and that modulation of ACE2 in coronavirus disease 2019 (COVID-19) drives severe organ injury. Methods: A series of NOTA-conjugated peptides derived from the known ACE2 inhibitor DX600 were synthesized, with variable linker identity. Since DX600 bears 2 cystine residues, both linear and cyclic peptides were studied. An ACE2 inhibition assay was used to identify lead compounds, which were labeled with ⁶⁸Ga to generate peptide radiotracers (⁶⁸Ga-NOTA-PEP). The aminocaproate-derived radiotracer ⁶⁸Ga-NOTA-PEP4 was subsequently studied in a humanized ACE2 (hACE2) transgenic model. Results: Cyclic DX-600-derived peptides had markedly lower half-maximal inhibitory concentrations than their linear counterparts. The 3 cyclic peptides with triglycine, aminocaproate, and polyethylene glycol linkers had calculated half-maximal inhibitory concentrations similar to or lower than the parent DX600 molecule. Peptides were readily labeled with ⁶⁸Ga, and the biodistribution of ⁶⁸Ga-NOTA-PEP4 was determined in an hACE2 transgenic murine cohort. Pharmacologic concentrations of coadministered NOTA-PEP (blocking) showed a significant reduction of ⁶⁸Ga-NOTA-PEP4 signals in the heart, liver, lungs, and small intestine. Ex vivo hACE2 activity in these organs was confirmed as a correlate to in vivo results. Conclusion: NOTA-conjugated cyclic peptides derived from the known ACE2 inhibitor DX600 retain their activity when N-conjugated for ⁶⁸Ga chelation. In vivo studies in a transgenic hACE2 murine model using the lead tracer, ⁶⁸Ga-NOTA-PEP4, showed specific binding in the heart, liver, lungs and intestine-organs known to be affected in SARS-CoV-2 infection. These results suggest that ⁶⁸Ga-NOTA-PEP4 could be used to detect organ-specific suppression of ACE2 in SARS-CoV-2-infected murine models and COVID-19 patients.

Background

Immune reconstitution inflammatory syndrome (IRIS) is a phenomenon initially described in patients with human immunodeficiency virus. Upon initiation of combination antiretroviral therapy, recovery of cellular immunity triggers inflammation to a preexisting infection or antigen that causes paradoxical worsening of clinical disease. A similar phenomenon can occur in human immunodeficiency virus-negative patients, including pregnant women, neutropenic hosts, solid-organ or stem cell transplant recipients, and patients receiving tumor necrosis factor inhibitors.

Observations

We report a case of leprosy unmasking and downgrading reaction after stem cell transplantation that highlights some of the challenges inherent to the diagnosis of IRIS, especially in patients without human immunodeficiency virus infection, as well as review the spectrum of previously reported cases of IRIS reactions in this population.

Conclusions

The mechanism of immune reconstitution reactions is complex and variable, depending on the underlying antigen and the mechanism of immunosuppression or shift in immune status. Use of the term IRIS can aid our recognition of an important phenomenon that occurs in the setting of immunosuppression or shifts in immunity but should not deter us from thinking critically about the distinct processes that underlie this heterogeneous group of conditions.

Increased mortality rates from infectious diseases is a growing public health concern. Successful management of acute bacterial infections requires early diagnosis and treatment, which are not always easy to achieve. Structural imaging techniques such as CT and MRI are often applied to this problem. However, these methods generally rely on secondary inflammatory changes and are frequently not specific to infection. The use of nuclear medicine techniques can add crucial complementary information, allowing visualization of infectious pathophysiology beyond morphologic imaging. This review will discuss the current structural and functional imaging techniques used for the diagnosis of bacterial infection and their roles in different clinical scenarios. We will also present several new radiotracers in development, with an emphasis on probes targeting bacteria-specific metabolism. As highlighted by the current coronavirus disease 2019 epidemic, caused by the novel severe acute respiratory syndrome coronavirus 2, similar thinking may apply in imaging viral pathogens; for this case, prominent effects on host proteins, most notably angiotensin-converting enzyme 2, might also provide worthwhile imaging targets.

WOPR-domain proteins are found throughout the fungal kingdom where they function as master regulators of cell morphology and pathogenesis. Genetic and biochemical experiments previously demonstrated that these proteins bind to specific DNA sequences and thereby regulate transcription. However, their primary sequence showed no relationship to any known DNA-binding domain, and the basis for their ability to recognize DNA sequences remained unknown. Here, we describe the 2.6-Å crystal structure of a WOPR domain in complex with its preferred DNA sequence. The structure reveals that two highly conserved regions, separated by an unconserved linker, form an interdigitated β-sheet that is tilted into the major groove of DNA. Although the main interaction surface is in the major groove, the highest-affinity interactions occur in the minor groove, primarily through a deeply penetrating arginine residue. The structure reveals a new, unanticipated mechanism by which proteins can recognize specific sequences of DNA.

This review highlights recent efforts to detect bacteria using engineered small molecules that are processed and incorporated similarly to their natural counterparts. There are both scientific and clinical justifications for these endeavors. The use of detectable, cell-wall targeted chemical probes has elucidated microbial behavior, with several fluorescent labeling methods in widespread laboratory use. Furthermore, many existing efforts including ours, focus on developing new imaging tools to study infection in clinical practice. The bacterial cell wall, a remarkably rich and complex structure, is an outstanding target for bacteria-specific detection. Several cell wall components are found in bacteria but not mammals, especially peptidoglycan, lipopolysaccharide, and teichoic acids. As this review highlights, the development of laboratory tools for fluorescence microscopy has vastly outstripped related positron emission tomography (PET) or single photon emission computed tomography (SPECT) radiotracer development. However, there is great synergy between these chemical strategies, which both employ mimicry of endogenous substrates to incorporate detectable structures. As the field of bacteria-specific imaging grows, it will be important to understand the mechanisms involved in microbial incorporation of radionuclides. Additionally, we will highlight the clinical challenges motivating this imaging effort.

The ESX (or Type VII) secretion systems are protein export systems in mycobacteria and many Gram-positive bacteria that mediate a broad range of functions including virulence, conjugation, and metabolic regulation. These systems translocate folded dimers of WXG100-superfamily protein substrates across the cytoplasmic membrane. We report the cryo-electron microscopy structure of an ESX-3 system, purified using an epitope tag inserted with recombineering into the chromosome of the model organism Mycobacterium smegmatis. The structure reveals a stacked architecture that extends above and below the inner membrane of the bacterium. The ESX-3 protomer complex is assembled from a single copy of the EccB₃, EccC₃, and EccE₃ and two copies of the EccD₃ protein. In the structure, the protomers form a stable dimer that is consistent with assembly into a larger oligomer. The ESX-3 structure provides a framework for further study of these important bacterial transporters.

SummaryMycobacterium tuberculosis and Staphylococcus aureus secrete virulence factors via Type VII protein secretion (T7S), a system that intriguingly requires all of its secretion substrates for activity. To gain insights into T7S function, we used structural approaches to guide studies of the putative translocase EccC, a unique enzyme with three ATPase domains, and its secretion substrate EsxB. The crystal structure of EccC revealed that the ATPase domains are joined by linker/pocket interactions that modulate its enzymatic activity. EsxB binds via its signal sequence to an empty pocket on the C-terminal ATPase domain, which is accompanied by an increase in ATPase activity. Surprisingly, substrate binding does not activate EccC allosterically, but rather by stimulating its multimerization. Thus, the EsxB substrate is also an integral T7S component, illuminating a mechanism that helps explain interdependence of substrates and suggests a model in which binding of substrates modulates their coordinate release from the bacterium.

Chlamydia trachomatis is an obligate intracellular pathogen that resides in a membrane-bound compartment, the inclusion. The bacteria secrete a unique class of proteins, Incs, which insert into the inclusion membrane and modulate the host-bacterium interface. We previously reported that IncE binds specifically to the Sorting Nexin 5 Phox domain (SNX5-PX) and disrupts retromer trafficking. Here, we present the crystal structure of the SNX5-PX:IncE complex, showing IncE bound to a unique and highly conserved hydrophobic groove on SNX5. Mutagenesis of the SNX5-PX:IncE binding surface disrupts a previously unsuspected interaction between SNX5 and the cation-independent mannose-6-phosphate receptor (CI-MPR). Addition of IncE peptide inhibits the interaction of CI-MPR with SNX5. Finally, C. trachomatis infection interferes with the SNX5:CI-MPR interaction, suggesting that IncE and CI-MPR are dependent on the same binding surface on SNX5. Our results provide new insights into retromer assembly and underscore the power of using pathogens to discover disease-related cell biology.