Mechanisms of Salmonella Typhi Host Restriction.

Salmonella enterica serovar Typhi (S. Typhi) is the cause of typhoid fever, a life-threatening bacterial infection that is very common in the developing world. Recent spread of antimicrobial resistant isolates of S. Typhi makes typhoid fever, a global public health risk. Despite being a common disease, still very little is known about the molecular mechanisms underlying typhoid fever and S. Typhi pathogenesis. In contrast to other Salmonellae, S. Typhi can only infect humans. The molecular bases of this human restriction are mostly unknown. Recent studies identified a novel pathway that contributes to S. Typhi human restriction and is required for killing S. Typhi in macrophages of nonsusceptible species. The small Rab GTPase Rab32 and its guanine nucleotide exchange factor BLOC-3 are the critical components of this pathway. These proteins were already well known as important regulators of intracellular membrane transport. In particular, they are central for the transport of enzymes that synthetize melanin in pigment cells. The recent findings that Rab32 and BLOC-3 are required for S. Typhi host restriction point out to a novel mechanism restricting the growth of bacterial pathogen, dependent on the transport of still unknown molecule(s) to the S. Typhi vacuole. The identification of this novel antimicrobial pathway constitutes a critical starting point to study molecular mechanisms killing bacterial pathogens and possibly identify novel antimicrobial molecules.

Host restriction in Salmonella: insights from Rab GTPases.

Salmonella enterica is a foodborne intracellular pathogen that can invade intestinal epithelial cells and survive in macrophages of susceptible hosts. Although belonging to the same species, individual Salmonella enterica serovars behave as very different pathogens. Indeed, they can cause very different diseases (from mild gastroenteritis to deadly systemic diseases) and have distinctive host selectivity. Salmonella enterica serovars Typhi (S. Typhi) is a unique serovar that has evolved to infect only humans and cause typhoid fever, a life-threatening systemic disease killing more than 200,000 people every year. The mechanisms that make S. Typhi able to infect only humans are mostly unknown. Recently, an antimicrobial traffic pathway dependent on the Rab GTPase Rab32 and its exchange factor BLOC-3 was found to be critical to kill S. Typhi in macrophages from non-susceptible hosts, suggesting that this pathway delivers an antimicrobial factor to the S. Typhi vacuole. Here we discuss this finding in the light of the current knowledge of pathogen killing mechanisms.

Structural and enzymatic characterization of a host-specificity determinant from Salmonella.

GtgE is an effector protein from Salmonella Typhimurium that modulates trafficking of the Salmonella-containing vacuole. It exerts its function by cleaving the Rab-family GTPases Rab29, Rab32 and Rab38, thereby preventing the delivery of antimicrobial factors to the bacteria-containing vacuole. Here, the crystal structure of GtgE at 1.65 Šresolution is presented, and structure-based mutagenesis and in vivo infection assays are used to identify its catalytic triad. A panel of cysteine protease inhibitors were examined and it was determined that N-ethylmaleimide, antipain and chymostatin inhibit GtgE activity in vitro. These findings provide the basis for the development of novel therapeutic strategies to combat Salmonella infections.

Proteolytic targeting of Rab29 by an effector protein distinguishes the intracellular compartments of human-adapted and broad-host Salmonella.

Unlike broad-host Salmonella serovars, which cause self-limiting disease, Salmonella enterica serovar Typhi can infect only humans causing typhoid fever, a life-threatening systemic disease. The molecular bases for these differences are presently unknown. Here we show that the GTPase Rab29 (Rab7L1) distinguishes the intracellular vacuole of human-adapted and broad-host Salmonella serovars. A screen to identify host factors required for the export of typhoid toxin, which is exclusively encoded by the human-specific Salmonella enterica serovars Typhi (S. Typhi) and Paratyphi (S. Paratyphi) identified Rab29. We found that Rab29 is recruited to the S. Typhi-containing vacuole but not to vacuoles containing broad-host Salmonella. We observed that in cells infected with broad-host Salmonella Rab29 is specifically cleaved by the proteolytic activity of GtgE, a unique type III secretion effector protein that is absent from S. Typhi. An S. Typhi strain engineered to express GtgE and therefore able to cleave Rab29 exhibited increased intracellular replication in human macrophages. These findings indicate significant differences in the intracellular biology of human-adapted and broad-host Salmonella and show how subtle differences in the assortment of effector proteins encoded by highly related pathogens can have a major impact in their biology.

CtBP3/BARS drives membrane fission in dynamin-independent transport pathways.

Membrane fission is a fundamental step in membrane transport. So far, the only fission protein machinery that has been implicated in in vivo transport involves dynamin, and functions in several, but not all, transport pathways. Thus, other fission machineries may exist. Here, we report that carboxy-terminal binding protein 3/brefeldin A-ribosylated substrate (CtBP3/BARS) controls fission in basolateral transport from the Golgi to the plasma membrane and in fluid-phase endocytosis, whereas dynamin is not involved in these steps. Conversely, CtBP3/BARS protein is inactive in apical transport to the plasma membrane and in receptor-mediated endocytosis, both steps being controlled by dynamin. This indicates that CtBP3/BARS controls membrane fission in endocytic and exocytic transport pathways, distinct from those that require dynamin.

A Small-Scale shRNA Screen in Primary Mouse Macrophages Identifies a Role for the Rab GTPase Rab1b in Controlling Salmonella Typhi Growth.

Salmonella Typhi is a human-restricted bacterial pathogen that causes typhoid fever, a life-threatening systemic infection. A fundamental aspect of S. Typhi pathogenesis is its ability to survive in human macrophages but not in macrophages from other animals (i.e. mice). Despite the importance of macrophages in establishing systemic S. Typhi infection, the mechanisms that macrophages use to control the growth of S. Typhi and the role of these mechanisms in the bacterium's adaptation to the human host are mostly unknown. To facilitate unbiased identification of genes involved in controlling the growth of S. Typhi in macrophages, we report optimized experimental conditions required to perform loss-of function pooled shRNA screens in primary mouse bone-marrow derived macrophages. Following infection with a fluorescent-labeled S. Typhi, infected cells are sorted based on the intensity of fluorescence (i.e. number of intracellular fluorescent bacteria). shRNAs enriched in the fluorescent population are identified by next-generation sequencing. A proof-of-concept screen targeting the mouse Rab GTPases confirmed Rab32 as important to restrict S. Typhi in mouse macrophages. Interestingly and rather unexpectedly, this screen also revealed that Rab1b controls S. Typhi growth in mouse macrophages. This constitutes the first report of a Rab GTPase other than Rab32 involved in S. Typhi host-restriction. The methodology described here should allow genome-wide screening to identify mechanisms controlling the growth of S. Typhi and other intracellular pathogens in primary immune cells.

The Rab32/BLOC-3-dependent pathway mediates host defense against different pathogens in human macrophages.

Macrophages provide a first line of defense against microorganisms, and while some mechanisms to kill pathogens such as the oxidative burst are well described, others are still undefined or unknown. Here, we report that the Rab32 guanosine triphosphatase and its guanine nucleotide exchange factor BLOC-3 (biogenesis of lysosome-related organelles complex-3) are central components of a trafficking pathway that controls both bacterial and fungal intracellular pathogens. This host-defense mechanism is active in both human and murine macrophages and is independent of well-known antimicrobial mechanisms such as the NADPH (reduced form of nicotinamide adenine dinucleotide phosphate)-dependent oxidative burst, production of nitric oxide, and antimicrobial peptides. To survive in human macrophages, Salmonella Typhi actively counteracts the Rab32/BLOC-3 pathway through its Salmonella pathogenicity island-1-encoded type III secretion system. These findings demonstrate that the Rab32/BLOC-3 pathway is a novel and universal host-defense pathway and protects mammalian species from various pathogens.

A novel pathway for exotoxin delivery by an intracellular pathogen.

Fundamental to the biology of many bacterial pathogens are bacterial proteins with the capacity to modulate host cellular functions. These bacterial proteins are delivered to the host's molecular targets by a great diversity of mechanisms of varying complexity. The different delivery mechanisms are adapted to the specific biology of the pathogen. Here we focus our attention on a recently described delivery pathway adapted to the biology of an intracellular pathogen, in which an exotoxin is delivered from an intracellular location to its molecular target through autocrine and paracrine pathways.

VARP and Rab9 Are Dispensable for the Rab32/BLOC-3 Dependent Salmonella Killing.

Salmonella enterica serovar Typhi (S. Typhi) is the causative agent of typhoid fever, a disease that kills an estimated 200,000 people annually. Previously, we discovered an antimicrobial pathway dependent on Rab32 and BLOC-3 (BRAM) that is critical to kill S. Typhi in murine macrophages. The BLOC-3 complex is comprised of the two sub-units HPS1 and HPS4 and exhibits guanine-nucleotide exchange factor (GEF) activity to Rab32. In melanocytes, Rab9 has been shown to interact with HPS4 and RUTBC1, a Rab32 GTPase activating (GAP) protein, and regulate the Rab32-mediated melanosome biogenesis. Intriguingly, Rab9-deficient melanocytes exhibit hypopigmentation, a similar phenotype to Rab32 or BLOC-3 deficient melanocytes. Additionally, VPS9-ankyrin-repeat-protein (VARP) has been shown to regulate melanocytic enzyme trafficking into the melanosomes through interaction with Rab32. Although Rab32, Rab9 and VARP are a part of melanogenesis in melanocytes, whether Rab9 and VARP are required for the BRAM mediated killing in macrophages is currently unknown. Here we showed that HPS4 is recruited to the Salmonella-containing vacuoles (SCV) and over-expression of BLOC-3 significantly increased Rab32-positive bacteria vacuoles. We found that SCV acquire Rab9, however over-expressing Rab9 did not change HPS4 localization on bacteria vacuoles. Importantly, we used shRNA to knock-down Rab9 and VARP in macrophages and showed that these proteins are dispensable for Rab32 recruitment to the SCV. Furthermore, we assessed the survival of S. Typhimurium in macrophages deficient for Rab9 or VARP and demonstrated that these proteins are not essential for BRAM pathway-dependent killing.

A technical skills elective program for pre-clerkship medical students reduces levels of high anxiety for performing technical skills.

BACKGROUND: We investigated the effect of a simulation-based technical skills course on rates of high anxiety reported by pre-clerkship medical students for basic and advanced technical skills. METHODS: Twenty-two second year medical students reported levels of anxiety by electronic survey for 21 technical skills before and after the course. A peer group of 75 students were invited to complete the survey for comparison. RESULTS: We received 21 (95.5%) responses before and after the course, and 12 (57.1%) in a three-month follow-up. Rates of high anxiety ranged from 19 to 86% across skills before the course and 0-48% afterward. There was no statistically significant difference in high anxiety reported in a three-month follow-up survey. The rates of high anxiety reported were reduced across all skills for course participants compared to the responding peer group of 32 (42.7%), reaching a statistically significant difference for 15/21 skills (P < 0.05). CONCLUSIONS: Participation in this technical skills course was associated with decreased reports of high anxiety by pre-clerkship medical students regarding the performance of basic and advanced technical skills.

Mechanisms directing the nuclear localization of the CtBP family proteins.

The C-terminal binding protein (CtBP) family includes four proteins (CtBP1 [CtBP1-L], CtBP3/BARS [CtBP1-S], CtBP2, and RIBEYE) which are implicated both in transcriptional repression and in intracellular trafficking. However, the precise mechanisms by which different CtBP proteins are targeted to different subcellular regions remains unknown. Here, we report that the nuclear import of the various CtBP proteins and splice isoforms is differentially regulated. We show that CtBP2 contains a unique nuclear localization signal (NLS) located within its N-terminal region, which contributes to its nuclear accumulation. Using heterokaryon assays, we show that CtBP2 is capable of shuttling between the nucleus and cytoplasm of the cell. Moreover, CtBP2 can heterodimerize with CtBP1-L and CtBP1-S and direct them to the nucleus. This effect strongly depends on the CtBP2 NLS. PXDLS motif-containing transcription factors, such as BKLF, that bind CtBP proteins can also direct them to the nucleus. We also report the identification of a splice isoform of CtBP2, CtBP2-S, that lacks the N-terminal NLS and localizes to the cytoplasm. Finally, we show that mutation of the CtBP NADH binding site impairs the ability of the proteins to dimerize and to associate with BKLF. This reduces the nuclear accumulation of CtBP1. Our results suggest a model in which the nuclear localization of CtBP proteins is influenced by the CtBP2 NLS, by binding to PXDLS motif partner proteins, and through the effect of NADH on CtBP dimerization.

Taking control: Hijacking of Rab GTPases by intracellular bacterial pathogens.

Intracellular bacterial pathogens survive and replicate within specialized eukaryotic cell organelles. To establish their intracellular niches these pathogens have adopted sophisticated strategies to control intracellular membrane trafficking. Since Rab-family GTPases are critical regulators of endocytic and secretory membrane trafficking events, many intracellular pathogens have evolved specific mechanisms to modulate or hijack Rab GTPases dynamics and trafficking functions. One such strategy is the delivery of bacterial effectors through specialized machines to specifically target Rab GTPases. Some of these effectors functionally mimic host proteins that regulate the Rab GTP cycle, while others regulate Rabs proteins through their post-translation modifications or proteolysis. In this review, we examine how the localization and function of Rab-family GTPases are altered during infection with 3 well-studied intracellular bacterial pathogens, Mycobacterium tuberculosis, Salmonella enterica and Legionella pneumophila. We also discuss recent findings about specific mechanisms by which these intracellular pathogens target this protein family.

Rab32 restriction of intracellular bacterial pathogens.

Our immune system is engaged in a continuous battle against invading pathogens, many of which have evolved to survive in intracellular niches of mammalian hosts. A variety of cellular processes are involved in preventing bacterial invasion or in killing bacteria that successfully invade host cells. Recently, the Rab GTPase Rab32 emerged as critical regulator of a host defense pathway that can eliminate bacterial pathogens. Salmonella enterica is an intracellular bacterium and a major cause of infections and deaths in humans. Rab32 and its guanine nucleotide exchange factor BLOC-3 are essential to prevent the growth of the human-restricted Salmonella enterica serovar Typhi (S. Typhi) in mice, a non-susceptible host. The importance of the Rab32/BLOC-3 pathway has been recently confirmed by the finding that broad-host Salmonella enterica serovars deliver 2 bacterial effectors to neutralize this pathway and infect mice. Rab32 has also been shown to control infection by Listeria monocytogenes, another medically relevant intracellular pathogen. In addition, genetic evidence indicate a possible role of Rab32 in controlling leprosy, a disease caused by Mycobacterium leprae in humans, suggesting that a Rab32-dependent pathway can also act as a host defense pathway in humans. The Rab32 role in bacterial pathogen restriction is discussed here and compared to the function of this GTPase in other cellular processes.

The Surgical Skills and Technology Elective Program (SSTEP): A comprehensive simulation-based surgical skills initiative for preclerkship medical students.

BACKGROUND: The Surgical Skills and Technology Elective Program (SSTEP) is a voluntary preclerkship surgical bootcamp that uses simulation learning to build procedural knowledge and technical skills before clerkship. METHODS: Eighteen second year students (n = 18) participated in simulation workshops over the course of 7 days to learn clerkship-level procedural skills. A manual was supplied with the program outline. Assessment of the participants involved: 1) a written exam 2) a single videotaped Objective Structured Assessment of Technical Skill (OSATS) station 3) an exit survey to document changes in career choices. RESULTS: Compared to the mean written pre-test score students scored significantly higher on the written post-test (35.83 ± 6.56 vs. 52.11 ± 5.95 out of 73) (p = 0.01). Technical skill on the OSATS station demonstrated improved performance and confidence following the program (10.10 vs. 17.94 out of 25) (p = 0.05). Most participants (72%) re-considered their choices of surgical electives. CONCLUSIONS: A preclerkship surgical skills program not only stimulates interest in surgery but can also improve surgical knowledge and technical skills prior to clerkship.

The C-terminal domain of the transcriptional corepressor CtBP is intrinsically unstructured.

C-terminal binding proteins (CtBPs) are moonlighting proteins involved in nuclear transcriptional corepression and in Golgi membrane tubule fission. Structural information on CtBPs is available for their substrate-binding domain, responsible for transcriptional repressor recognition/binding, and for the nucleotide-binding domain, involved in NAD(H)-binding and dimerization. On the contrary, little is known about the structure of CtBP C-terminal region ( approximately 90 residues), hosting sites for post-translational modifications. In the present communication we apply a combined approach based on bioinformatics, nuclear magnetic resonance, circular dichroism spectroscopy, and small-angle X-ray scattering, and we show that the CtBP C-terminal region is intrinsically unstructured in the full-length CtBP and in constructs lacking the substrate- and/or the nucleotide-binding domains. The flexible nature of this protein region, and its structural transitions, may be instrumental for CtBP recognition and binding to diverse molecular partners.

A Bacterial Pathogen Targets a Host Rab-Family GTPase Defense Pathway with a GAP.

Cell-autonomous defense mechanisms are potent strategies that protect individual cells against intracellular pathogens. The Rab-family GTPase Rab32 was previously shown to restrict the intracellular human pathogen Salmonella Typhi, but its potential broader role in antimicrobial defense remains unknown. We show that Rab32 represents a general cell-autonomous, antimicrobial defense that is counteracted by two Salmonella effectors. Mice lacking Rab-32 or its nucleotide exchange factor BLOC-3 are permissive to S. Typhi infection and exhibit increased susceptibility to S. Typhimurium. S. Typhimurium counters this defense pathway by delivering two type III secretion effectors, SopD2, a Rab32 GAP, and GtgE, a specific Rab32 protease. An S. Typhimurium mutant strain lacking these two effectors exhibits markedly reduced virulence, which is fully restored in BLOC-3-deficient mice. These results demonstrate that a cell-autonomous, Rab32-dependent host defense pathway plays a central role in the defense against vacuolar pathogens and describe a mechanism evolved by a bacterial pathogen to counter it.

Does Bronchoscopic Evaluation of Inhalation Injury Severity Predict Outcome?

Although fiber-optic bronchoscopy is essential in the diagnosis of smoke inhalation injury (INH), controversy still exists over whether or not the visualized severity of the mucosal injury predicts clinically meaningful outcomes. The purpose of this study was to assess whether the grade of mucosal INH severity was associated with various outcomes among adult burn patients. We conducted a retrospective review of all patients requiring greater than or equal to 48 hours of mechanical ventilation who were admitted between January 1, 2007 and June 1, 2014 to an adult regional American Burn Association-verified burn center. Bronchoscopy was performed on all subjects at burn center admission and grading of severity was documented using the grades 0 to 4 abbreviated injury score (AIS). Subjects with grade 1 or 2 injury formed the low-grade INH group, whereas those with grade 3 or 4 injury formed the high-grade INH group. Values are shown as the median (first to third quartiles). A P value less than .05 was considered significant. The study population consisted of 160 subjects (age, 48 [35-60] years; %TBSA burn, 28 [19-39.9]; % full thickness burn, 12.8 [0-30]; and 61% with INH). There were no significant differences in age, %TBSA burn, or % full thickness burn between subjects with different individual INH severity grades. Oxygenation on the day of injury worsened significantly as the severity of INH increased, but otherwise there were no significant differences in 24 and 48-hour fluid requirements, duration of ventilation, ventilator free days, incidence of acute respiratory distress syndrome, or mortality between subjects with different individual grades of INH severity. Subjects with high-grade INH showed statistically insignificant trends toward larger 48-hour fluid volumes (P = .07), poorer oxygenation over the first 3 post burn days (P = .055), longer duration of ventilation (P = .08), and fewer ventilator free days (P = .047) than low-grade INH. High-grade and low-grade INH subjects did not differ significantly in the incidence of acute respiratory distress syndrome or mortality. The individual grades of the 0 to 4 AIS INH severity grading scale were not particularly robust in the prediction of various outcomes among a population of adult burn patients. However, clinically relevant trends toward worsened oxygenation over postburn days 0 to 3, longer duration of mechanical ventilation, and reduced ventilator-free days in association with more severe INH were identified when subjects were broadly stratified into low-grade (grades 1and 2) INH and high-grade (grades 3 and 4) INH. This suggests that there may clinically meaningful differences between patients with less and more severe INH, and that further refinement of the grades 0 to 4 AIS INH severity should be subjected to additional investigation.

The Acute Respiratory Distress Syndrome (ARDS) in mechanically ventilated burn patients: An analysis of risk factors, clinical features, and outcomes using the Berlin ARDS definition.

BACKGROUND: The Berlin definition of Acute Respiratory Distress Syndrome (ARDS) has been applied to military burns resulting from combat-related trauma, but has not been widely studied among civilian burns. This study's purpose was to use the Berlin definition to determine the incidence of ARDS, and its associated respiratory morbidity, and mortality among civilian burn patients. METHODS: Retrospective study of burn patients mechanically ventilated for ≥48h at an American Burn Association-verified burn center. The Berlin criteria identified patients with mild, moderate, and severe ARDS. Logistic regression was used to identify variables predictive of moderate to severe ARDS, and mortality. The outcome measures of interest were duration of mechanical ventilation and in-hospital mortality. Values are shown as the median (Q1-Q3). RESULTS: We included 162 subjects [24% female, age 48 (35-60), % total body surface area (TBSA) burn 28 (19-40), % body surface area (BSA) full thickness (FT) burn 13 (0-30), and 62% with inhalation injury]. The incidence of ARDS was 43%. Patients with ARDS had larger %TBSA burns [30.5 (23.1-47.0) vs. 24.8 (17.1-35), p=0.007], larger FT burns [20.5(5.4-35.5) vs. 7 (0-22.1), p=0.001], but had no significant difference in the incidence of inhalation injury (p=0.216), compared to those without ARDS. The % FT burn predicted the development of moderate to severe ARDS [OR 1.034, 95%CI (1.013-1.055), p=0.001]. ARDS developed in the 1st week after burn in 86% of cases. Worsening severity of ARDS was associated with increased days of mechanical ventilation in survivors (p=0.001), a reduction in ventilator-free days/1st 30 days in all subjects (p=0.004), and a strong indication of increased mortality (0% in mild ARDS vs. 50% in severe ARDS, unadjusted p=0.02). Neither moderate ARDS nor severe ARDS were significant predictors of death. CONCLUSIONS: ARDS is common among mechanically ventilated civilian burn patients, and develops early after burn. The extent of full thickness burn predicted development of moderate to severe ARDS. Increasing severity of ARDS based upon the Berlin definition was associated with a significantly greater duration of mechanical ventilation and a trend toward higher mortality.

Purification and functional properties of the membrane fissioning protein CtBP3/BARS.

The fissioning protein CtBP3/BARS is a member of the CtBP transcription corepressor family of proteins. The characterization of this fissioning activity of CtBP3/BARS in both isolated Golgi membranes and in intact cells has indicated that the CtBP family includes multifunctional proteins that can act both in the nucleus and in the cytoplasm. The fissiogenic activity of CtBP3/BARS has a role in the fragmentation of the Golgi complex during mitosis and during intracellular membrane transport. This was demonstrated using a number of approaches and reagents, which are discussed in the following text, and which include recombinant proteins and mutants, antibodies, protein overexpression, RNA interference, antisense oligonucleotides, cell permeabilization, and electron miscroscopy, together with biochemical assays such as that for ADP-ribosylation.

An examination of the mechanisms by which neural precursors augment recovery following spinal cord injury: a key role for remyelination.

The mechanisms by which neural precursor cells (NPCs) enhance functional recovery from spinal cord injury (SCI) remain unclear. Spinal cord injured rats were transplanted with wild-type mouse NPCs, shiverer NPCs unable to produce myelin, dead NPCs, or media. Most animals also received minocycline, cyclosporine, and perilesional infusion of trophins. Motor function was graded according to the BBB scale. H&E/LFB staining was used to assess gray and white matter, cyst, and lesional tissue. Mature oligodendrocytes and ED1(+) inflammatory cells were quantitated. Confocal and electron microscopy were used to assess the relationship between the transplanted cells and axons. Pharmacotherapy and trophin infusion preserved gray matter, white matter, and oligodendrocytes. Trophin infusion also significantly increased cyst and lesional tissue volume as well as inflammatory infiltrate, and functional recovery was reduced. Animals transplanted with wild-type NPCs showed greatest functional recovery; animals transplanted with shiverer NPCs performed the worst. Wild-type NPCs remyelinated host axons. Shiverer NPCs ensheathed axons but did not produce MBP. These results suggest that remyelination by NPCs is an important contribution to functional recovery following SCI. Shiverer NPCs may prevent remyelination by endogenous cells capable of myelin formation. These findings suggest that remyelination is an important therapeutic target following SCI.