Multisensory alarm to benefit alarm identification and decrease workload: a feasibility study.

The poor design of conventional auditory medical alarms has contributed to alarm desensitization, and eventually, alarm fatigue in medical personnel. This study tested a novel multisensory alarm system which aims to help medical personnel better interpret and respond to alarm annunciation during periods of high cognitive load such as those found within intensive care units. We tested a multisensory alarm that combined auditory and vibrotactile cues to convey alarm type, alarm priority, and patient identity. Testing was done in three phases: control (conventional auditory), Half (limited multisensory alarm), and Full (complete multisensory alarm). Participants (N = 19, undergraduates) identified alarm type, priority, and patient identity (patient 1 or 2) using conventional and multisensory alarms, while simultaneously completing a cognitively demanding task. Performance was based on reaction time (RT) and identification accuracy of alarm type and priority. Participants also reported their perceived workload. RT was significantly faster for the Control phase (p < 0.05). Participant performance in identifying alarm type, priority, and patient did not differ significantly between the three phase conditions (p = 0.87, 0.37, and 0.14 respectively). The Half multisensory phase produced the lowest mental demand, temporal demand, and overall perceived workload score. These data suggest that implementation of a multisensory alarm with alarm and patient information may decrease perceived workload without significant changes in alarm identification performance. Additionally, a ceiling effect may exist for multisensory stimuli, with only part of an alarm benefitting from multisensory integration.

High-resolution mapping reveals that microniches in the gastric glands control Helicobacter pylori colonization of the stomach.

Lifelong infection of the gastric mucosa by Helicobacter pylori can lead to peptic ulcers and gastric cancer. However, how the bacteria maintain chronic colonization in the face of constant mucus and epithelial cell turnover in the stomach is unclear. Here, we present a new model of how H. pylori establish and persist in stomach, which involves the colonization of a specialized microenvironment, or microniche, deep in the gastric glands. Using quantitative three-dimensional (3D) confocal microscopy and passive CLARITY technique (PACT), which renders tissues optically transparent, we analyzed intact stomachs from mice infected with a mixture of isogenic, fluorescent H. pylori strains with unprecedented spatial resolution. We discovered that a small number of bacterial founders initially establish colonies deep in the gastric glands and then expand to colonize adjacent glands, forming clonal population islands that persist over time. Gland-associated populations do not intermix with free-swimming bacteria in the surface mucus, and they compete for space and prevent newcomers from establishing in the stomach. Furthermore, bacterial mutants deficient in gland colonization are outcompeted by wild-type (WT) bacteria. Finally, we found that host factors such as the age at infection and T-cell responses control bacterial density within the glands. Collectively, our results demonstrate that microniches in the gastric glands house a persistent H. pylori reservoir, which we propose replenishes the more transient bacterial populations in the superficial mucosa.

Comparative quantitative clinical, neuroimaging, and functional profiles in children with acute flaccid myelitis at acute and convalescent stages of disease.

AIM: To quantify characteristics in acute flaccid myelitis (AFM) at acute and convalescent stages. METHOD: This was a retrospective case series of children with AFM evaluated at a single institution in the USA (2014-2017). Acute inflammatory/ischemic myelopathies were excluded. Neurological assessments and segmental quantitative analysis of signal abnormalities on magnetic resonance imaging (MRI) of the brain and spinal cord were performed. RESULTS: Sixteen patients (11 males, five females) were evaluated. Median age at onset was 4 years (interquartile range [IQR] 3-6y). All had parainfectious acute-onset limb weakness, lower motor neuron examination, and spinal fluid pleocytosis. On acute spinal cord MRI, longitudinally extensive T2 hyperintensities were identified throughout the spinal cord mostly within grey matter; five out of 12 patients had dorsal brainstem T2 hyperintensities. At a median of 2 months follow-up (IQR 2-3mo), spinal cord MRI improved in seven out of nine patients although focal T2 hyperintensities persisted in cervical and lumbar grey matter. At a median follow-up of 4 months (IQR 2-6mo), Medical Research Council sum score rose from a median of 29 to 32; distal muscle groups improved more than proximal ones; four out of 16 patients were ventilator-dependent; and two out of 16 patients were quadriplegic. INTERPRETATION: While patients may show marked improvement on neuroimaging from acute to convalescent stages, the majority of children with AFM have limited motor recovery and continued disability. Clinicians should consider the timing of clinical and neuroimaging exams when assessing diagnosis and prognosis. WHAT THIS PAPER ADDS: During the 2014 to 2017 acute flaccid myelitis outbreak in the USA, clinical recovery was better in distal than proximal muscle groups. Lumbar spinal cord showed more residual abnormalities at convalescence.

The type III secretion system apparatus determines the intracellular niche of bacterial pathogens.

Upon entry into host cells, intracellular bacterial pathogens establish a variety of replicative niches. Although some remodel phagosomes, others rapidly escape into the cytosol of infected cells. Little is currently known regarding how professional intracytoplasmic pathogens, including Shigella, mediate phagosomal escape. Shigella, like many other Gram-negative bacterial pathogens, uses a type III secretion system to deliver multiple proteins, referred to as effectors, into host cells. Here, using an innovative reductionist-based approach, we demonstrate that the introduction of a functional Shigella type III secretion system, but none of its effectors, into a laboratory strain of Escherichia coli is sufficient to promote the efficient vacuole lysis and escape of the modified bacteria into the cytosol of epithelial cells. This establishes for the first time, to our knowledge, a direct physiologic role for the Shigella type III secretion apparatus (T3SA) in mediating phagosomal escape. Furthermore, although protein components of the T3SA share a moderate degree of structural and functional conservation across bacterial species, we show that vacuole lysis is not a common feature of T3SA, as an effectorless strain of Yersinia remains confined to phagosomes. Additionally, by exploiting the functional interchangeability of the translocator components of the T3SA of Shigella, Salmonella, and Chromobacterium, we demonstrate that a single protein component of the T3SA translocon-Shigella IpaC, Salmonella SipC, or Chromobacterium CipC-determines the fate of intracellular pathogens within both epithelial cells and macrophages. Thus, these findings have identified a likely paradigm by which the replicative niche of many intracellular bacterial pathogens is established.

Functional relatedness in the Inv/Mxi-Spa type III secretion system family.

Type III Secretion Systems (T3SSs) are structurally conserved nanomachines that span the inner and outer bacterial membranes, and via a protruding needle complex contact host cell membranes and deliver type III effector proteins. T3SS are phylogenetically divided into several families based on structural basal body components. Here we have studied the evolutionary and functional conservation of four T3SS proteins from the Inv/Mxi-Spa family: a cytosolic chaperone, two hydrophobic translocators that form a plasma membrane-integral pore, and the hydrophilic 'tip complex' translocator that connects the T3SS needle to the translocon pore. Salmonella enterica serovar Typhimurium (S. Typhimurium), a common cause of food-borne gastroenteritis, possesses two T3SSs, one belonging to the Inv/Mxi-Spa family. We used invasion-deficient S. Typhimurium mutants as surrogates for expression of translocator orthologs identified from an extensive phylogenetic analysis, and type III effector translocation and host cell invasion as a readout for complementation efficiency, and identified several Inv/Mxi-Spa orthologs that can functionally substitute for the S. Typhimurium chaperone and translocator proteins. Functional complementation correlates with amino acid sequence identity between orthologs, but varies considerably between the four proteins. This is the first in-depth survey of the functional interchangeability of Inv/Mxi-Spa T3SS proteins acting directly at the host-pathogen interface.

Down's Syndrome and Triple Negative Breast Cancer: A Rare Occurrence of Distinctive Clinical Relationship.

Down's syndrome (DS), the most common genetic cause of significant intellectual disability in children and adults is caused by the trisomy of either all or a part of human chromosome 21 (HSA21). Patients with DS mostly suffer from characteristic tumor types. Although individual patients of DS are at a higher risk for acute leukemia and testicular cancers, other types of solid tumors including breast cancers are mostly uncommon and have significantly lower-than-expected age-adjusted incidence rates. Except for an increased risk of retinoblastomas, and lymphomas, the risk of developing solid tumors has been found to be lower in both children and adults, and breast cancer was found to be almost absent (Hasle H., The Lancet Oncology, 2001). A study conducted in the United States found only one death when 11.65 were expected (Scholl T et al., Dev Med Child Neurol. 1982). A recent study examined mammogram reports of women with DS treated in the largest medical facility specifically serving adults with DS in the United States. It was found that only 0.7% women with DS had been diagnosed with breast cancers (Chicoine B et al., Intellect Dev Disabil. 2015). Here we describe a case of breast cancer in a 25-year-old patient with DS. The disease was presented as lymph node positive carcinoma with alterations of tumor suppressor genes characteristic to the triple negative breast cancer subtype. Comprehensive Genomic Profiling (CGP) revealed a wild-type status for BRCA1. The CGP report showed a frameshift mutation, A359fs*10 of the tumor suppressor gene INPP4B and another frameshift mutation, R282fs*63 of tumor suppressor gene TP53 in the tumor biopsy as characteristically found in triple-negative breast cancers. The VUS (Variance of Unknown Significance) alteration(s) were identified in ASXL1 (L1395V), NTRK1 (G18E), DDR2 (I159T), RUNX1 (amplification), ERG (amplification), SOX2 (T26A), FAM123B (G1031D), and HNF1A (A301T). Bonafide cancer-related genes of chromosome 21 amplified in the patient's tumor are RUNX1 and ERG genes. After the completion of the radiation, the patient was placed on everolimus which was based on the result of her CGP report. Thus, post-mastectomy radiation therapy was completed with a recommendation for everolimus for one year. During the time of writing of this report, no metastatic lesions were identified. The patient currently has no evidence of disease.

Quantification of the functional expression of the Ca2+ -activated K+ channel KCa 3.1 on microglia from adult human neocortical tissue.

The KCa 3.1 channel (KCNN4) is an important modulator of microglia responses in rodents, but no information exists on functional expression on microglia from human adults. We isolated and cultured microglia (max 1% astrocytes, no neurons or oligodendrocytes) from neocortex surgically removed from epilepsy patients and employed electrophysiological whole-cell measurements and selective pharmacological tools to elucidate functional expression of KCa 3.1. The channel expression was demonstrated as a significant increase in the voltage-independent current by NS309, a KCa 3.1/KCa 2 activator, followed by full inhibition upon co-application with NS6180, a highly selective KCa 3.1 inhibitor. A major fraction (79%) of unstimulated human microglia expressed KCa 3.1, and the difference in current between full activation and inhibition (ΔKCa 3.1) was estimated at 292 ± 48 pA at -40 mV (n = 75), which equals at least 585 channels per cell. Serial KCa 3.1 activation/inhibition significantly hyperpolarized/depolarized the membrane potential. The isolated human microglia were potently activated by lipopolysaccharide (LPS) shown as a prominent increase in TNF-α production. However, incubation with LPS neither changed the KCa 3.1 current nor the fraction of KCa 3.1 expressing cells. In contrast, the anti-inflammatory cytokine IL-4 slightly increased the KCa 3.1 current per cell, but as the membrane area also increased, there was no significant change in channel density. A large fraction of the microglia also expressed a voltage-dependent current sensitive to the KCa 1.1 modulators NS1619 and Paxilline and an inward-rectifying current with the characteristics of a Kir channel. The high functional expression of KCa 3.1 in microglia from epilepsy patients accentuates the need for further investigations of its role in neuropathological processes. GLIA 2016;64:2065-2078.

A novel de novo dominant negative mutation in DNM1L impairs mitochondrial fission and presents as childhood epileptic encephalopathy.

DNM1L encodes dynamin-related protein 1 (DRP1/DLP1), a key component of the mitochondrial fission machinery that is essential for proper functioning of the mammalian brain. Previously reported probands with de novo missense mutations in DNM1L presented in the first year of life with severe encephalopathy and refractory epilepsy, with several dying within the first several weeks after birth. In contrast, we report identical novel missense mutations in DNM1L in two unrelated probands who experienced normal development for several years before presenting with refractory focal status epilepticus and subsequent rapid neurological decline. We expand the phenotype of DNM1L-related mitochondrial fission defects, reveal common unique clinical characteristics and imaging findings, and compare the cellular impact of this novel mutation to the previously reported A395D lethal variant. We demonstrate that our R403C mutation, which resides in the assembly region of DRP1, acts by a dominant-negative mechanism and reduces oligomerization, mitochondrial fission activity, and mitochondrial recruitment of DRP1, but to a lesser extent compared to the A395D mutation. In contrast to the initial report of neonatal lethality resulting from DNM1L mutation and DRP1 dysfunction, our results show that milder DRP1 impairment is compatible with normal early development and subsequently results in a distinct set of neurological findings. In addition, we identify a common pathogenic mechanism whereby DNM1L mutations impair mitochondrial fission. © 2016 Wiley Periodicals, Inc.

Pathogenic diversification of the gut commensal Providencia alcalifaciens via acquisition of a second type III secretion system.

Providencia alcalifaciens is a Gram-negative bacterium found in a wide variety of water and land environments and organisms. It has been isolated as part of the gut microbiome of animals and insects, as well as from stool samples of patients with diarrhea. Specific P. alcalifaciens strains encode gene homologs of virulence factors found in other pathogenic members of the same Enterobacterales order, such as Salmonella enterica serovar Typhimurium and Shigella flexneri. Whether these genes are also pathogenic determinants in P. alcalifaciens is not known. Here we have used P. alcalifaciens 205/92, a clinical isolate, with in vitro and in vivo infection models to investigate P. alcalifaciens -host interactions at the cellular level. Our particular focus was the role of two type III secretion systems (T3SS) belonging to the Inv-Mxi/Spa family. T3SS 1b is widespread in Providencia spp. and encoded on the chromosome. T3SS 1a is encoded on a large plasmid that is present in a subset of P. alcalifaciens strains, which are primarily isolates from diarrheal patients. Using a combination of electron and fluorescence microscopy and gentamicin protection assays we show that P. alcalifaciens 205/92 is internalized into eukaryotic cells, rapidly lyses its internalization vacuole and proliferates in the cytosol. This triggers caspase-4 dependent inflammasome responses in gut epithelial cells. The requirement for the T3SS 1a in entry, vacuole lysis and cytosolic proliferation is host-cell type specific, playing a more prominent role in human intestinal epithelial cells as compared to macrophages. In a bovine ligated intestinal loop model, P. alcalifaciens colonizes the intestinal mucosa, inducing mild epithelial damage with negligible fluid accumulation. No overt role for T3SS 1a or T3SS 1b was seen in the calf infection model. However, T3SS 1b was required for the rapid killing of Drosophila melanogaster . We propose that the acquisition of two T3SS by horizontal gene transfer has allowed P. alcalifaciens to diversify its host range, from a highly virulent pathogen of insects to an opportunistic gastrointestinal pathogen of animals.

Vaccination with a UV-irradiated genetically attenuated mutant of Staphylococcus aureus provides protection against subsequent systemic infection.

Staphylococcus aureus are gram-positive bacteria that cause clinically significant infections in humans. Severe S. aureus infections are particularly problematic in hospitalized patients and reoccur despite therapeutic measures. The absence of natural protective immune responses and the lack of high-throughput approaches to identify S. aureus antigens have imposed constraints in the development of effective vaccines. Here, we showed that vaccination with the genetically attenuated S. aureus mutant, inactivated using UV irradiation rather than heat, significantly increased survival and diminished bacterial burden and kidney abscesses when mice were challenged with virulent methicillin-sensitive or methicillin-resistant S. aureus. Protection conferred by immunization could be transferred to the naive host and was not observed in B-cell-deficient mice. Using a novel S. aureus whole-proteome microarray, we show that immunoglobulin G antibody responses to 83 proteins were observed in the immunized mice. These results suggest that protection against S. aureus infections requires antibody responses to the wide repertoire of antigens/virulence factors. Vaccination using UV-irradiated genetically attenuated S. aureus induces humoral immunity and provides a vaccine strategy for pathogens that fail to induce protective immunity. We also describe a novel, high-throughput technology to easily identify S. aureus antigens for vaccine development.

Measurement of Salmonella enterica Internalization and Vacuole Lysis in Epithelial Cells.

Establishment of an intracellular niche within mammalian cells is key to the pathogenesis of the gastrointestinal bacterium, Salmonella enterica serovar Typhimurium (S. Typhimurium). Here we will describe how to study the internalization of S. Typhimurium into human epithelial cells using the gentamicin protection assay. The assay takes advantage of the relatively poor penetration of gentamicin into mammalian cells; internalized bacteria are effectively protected from its antibacterial actions. A second assay, the chloroquine (CHQ) resistance assay, can be used to determine the proportion of internalized bacteria that have lysed or damaged their Salmonella-containing vacuole and are therefore residing within the cytosol. Its application to the quantification of cytosolic S. Typhimurium in epithelial cells will also be presented. Together, these protocols provide an inexpensive, rapid, and sensitive quantitative measure of bacterial internalization and vacuole lysis by S. Typhimurium.

Toward Inclusivity in Preclinical Drug Development: A Proposition to Start with Intestinal Organoids.

Representation of humans from diverse backgrounds in the drug development process is key to advancing health equity, and while clinical trial design has recently made strides toward greater inclusivity, preclinical drug development has struggled to make those same gains. One barrier to inclusion is the current lack of robust and established in vitro model systems that simultaneously capture the complexity of human tissues while representing patient diversity. Here, the use of primary human intestinal organoids as a mechanism to advance inclusive preclinical research is proposed. This in vitro model system not only recapitulates tissue functions and disease states, but also retains the genetic identity and epigenetic signatures of the donors from which they are derived. Thus, intestinal organoids are an ideal in vitro prototype for capturing human diversity. In this perspective, the authors call for an industry-wide effort to leverage intestinal organoids as a starting point to actively and intentionally incorporate diversity into preclinical drug programs.

Suspended hydrogel culture as a method to scale up intestinal organoids.

Primary tissue-derived epithelial organoids are a physiologically relevant in vitro intestinal model that have been implemented for both basic research and drug development applications. The existing method of culturing intestinal organoids in surface-attached native extracellular matrix (ECM) hydrogel domes is not readily amenable to large-scale culture and contributes to culture heterogeneity. We have developed a method of culturing intestinal organoids within suspended basement membrane extract (BME) hydrogels of various geometries, which streamlines the protocol, increases the scalability, enables kinetic sampling, and improves culture uniformity without specialized equipment or additional expertise. We demonstrate the compatibility of this method with multiple culture formats, and provide examples of suspended BME hydrogel organoids in downstream applications: implementation in a medium-throughput drug screen and generation of Transwell monolayers for barrier evaluation. The suspended BME hydrogel culture method will allow intestinal organoids, and potentially other organoid types, to be used more widely and at higher throughputs than previously possible.

A vector-encoded bispecific killer engager to harness virus-activated NK cells as anti-tumor effectors.

Treatment with oncolytic measles vaccines (MV) elicits activation of immune cells, including natural killer (NK) cells. However, we found that MV-activated NK cells show only modest direct cytotoxic activity against tumor cells. To specifically direct NK cells towards tumor cells, we developed oncolytic measles vaccines encoding bispecific killer engagers (MV-BiKE) targeting CD16A on NK cells and carcinoembryonic antigen (CEA) as a model tumor antigen. MV-BiKE are only slightly attenuated compared to parental MV and mediate secretion of functional BiKE from infected tumor cells. We tested MV-BiKE activity in cocultures of colorectal or pancreatic cancer cells with primary human NK cells. MV-BiKE mediate expression of effector cytokines, degranulation and specific anti-tumor cytotoxicity by NK cells. Experiments with patient-derived pancreatic cancer cultures indicate that efficacy of MV-BiKE may vary between individual tumors with differential virus permissiveness. Remarkably, we confirmed MV-BiKE activity in primaryhuman colorectal carcinoma specimens with autochthonous tumor and NK cells.This study provides proof-of-concept for MV-BiKE as a novel immunovirotherapy to harness virus-activated NK cells as anti-tumor effectors.

A Favorable Treatment Outcome in RANBP2 and Influenza Associated Acute Necrotizing Encephalitis.

In current literature, there is uncertainty in the pathophysiology and management of influenza-associated Acute Necrotizing Encephalitis. Because of this and the rarity of the disease, no clear treatment guidelines exist. It is thought that treatment after 24 h of symptom onset or known brainstem involvement are poor predictors of outcome. Here, we present a case that provides support for aggressive management of the inflammatory cascade with combination high-dose steroid, immunoglobulin, and anti-viral therapy with oseltamivir despite initiation after 24 h from symptom onset, brainstem involvement, and a pathogenic RANBP2 gene mutation which mechanistically increases oxidative stress, cytokine effects, and possibly viral invasion into brain tissue and vasculature.

Characterization of AN6001, a positive allosteric modulator of α6ß2-containing nicotinic acetylcholine receptors.

α6ß2-Containing nicotinic acetylcholine receptors (α6ß2* nAChRs) are predominantly expressed in midbrain dopaminergic neurons, including substantia nigra pars compacta (SNc) neurons and their projections to striatal regions, where they regulate dopamine release and nigrostriatal activity. It is well established that nAChR agonists exert protection against dopaminergic neurotoxicity in cellular assays and parkinsonian animal models. Historically, drug development in the nAChR field has been mostly focused on development of selective agonists and positive allosteric modulators (PAMs) for the predominant neuronal nAChRs, α7 and α4ß2. Here, we report the discovery and characterization of AN6001, a novel selective α6ß2* nAChR PAM. AN6001 mediated increases in both nicotine potency and efficacy at the human α6/α3ß2ß3V9'S nAChR in HEK293 cells, and it positively modulated ACh-evoked currents through both α6/α3ß2ß3V9'S and a concatenated ß3-α6-ß2-α6-ß2 receptor in Xenopus oocytes, displaying EC50 values of 0.58 µM and 0.40 µM, respectively. In contrast, the compound did not display significant modulatory activity at α4ß2, α3ß4, α7 and muscle nAChRs. AN6001 also increased agonist-induced dopamine release from striatal synaptosomes and augmented agonist-induced global cellular responses and inward currents in dopaminergic neurons in SNc slices (measured by Ca2+ imaging and patch clamp recordings, respectively). Finally, AN6001 potentiated the neuroprotective effect of nicotine at MPP+-treated primary dopaminergic neurons. Overall, our studies demonstrate the existence of allosteric sites on α6ß2* nAChRs and that positive modulation of native α6ß2* receptors strengthens DA signaling. Hence, AN6001 represents an important tool for studies of α6ß2* nAChRs and furthermore underlines the therapeutic potential in these receptors in Parkinson's disease.

Characterization of AN317, a novel selective agonist of α6ß2-containing nicotinic acetylcholine receptors.

Neuronal nicotinic acetylcholine receptors (nAChRs) are crucial mediators of central presynaptic, postsynaptic, and extrasynaptic signaling, and they are implicated in a range of CNS disorders. The numerous nAChR subtypes are differentially expressed and mediate distinct functions throughout the CNS, and thus there is considerable interest in developing subtype-selective nAChR modulators, both for use as pharmacological tools and as putative therapeutics. α6ß2-containing (α6ß2*) nAChRs are highly expressed in and regulate the activity of midbrain dopaminergic neurons, which makes them attractive drug targets in several psychiatric and neurological diseases, including nicotine addiction and Parkinson's disease. This paper presents the preclinical characterization of AN317, a novel α6ß2* agonist exhibiting functional selectivity toward other nAChRs, including α4ß2, α3ß4 and α7 receptors. AN317 induced [3H]dopamine release from rat striatal synaptosomes and augmented dopaminergic neuron activity in substantia nigra pars compacta brain slices in Ca2+ imaging and electrophysiological assays. In line with this, AN317 alleviated the high-frequency tremors arising from reserpine-mediated dopamine depletion in rats. Finally, AN317 mediated significant protective effects on cultured rat mesencephalic neurons treated with the dopaminergic neurotoxin MPP+. AN317 displays good bioavailability and readily crosses the blood-brain barrier, which makes it a unique tool for both in vitro and in vivo studies of native α6ß2* receptors in the nigrostriatal system and other dopaminergic pathways. Altogether, these findings highlight the potential of selective α6ß2* nAChR activation as a treatment strategy for symptoms and possibly even deceleration of disease progression in neurodegenerative diseases such as Parkinson's disease.

Measurement of Salmonella enterica Internalization and Vacuole Lysis in Epithelial Cells.

Establishment of an intracellular niche within mammalian cells is key to the pathogenesis of the gastrointestinal bacterium, Salmonella enterica serovar Typhimurium (S. Typhimurium). Here we will describe how to study the internalization of S. Typhimurium into human epithelial cells using the gentamicin protection assay. The assay takes advantage of the relatively poor penetration of gentamicin into mammalian cells; internalized bacteria are effectively protected from its antibacterial actions. A second assay, the chloroquine (CHQ) resistance assay, can be used to determine the proportion of internalized bacteria that have lysed or damaged their Salmonella-containing vacuole and are therefore residing within the cytosol. Its application to the quantification of cytosolic S. Typhimurium in epithelial cells will also be presented. Together, these protocols provide an inexpensive, rapid and sensitive quantitative measure of bacterial internalization and vacuole lysis by S. Typhimurium.

Controlled Activity of the Salmonella Invasion-Associated Injectisome Reveals Its Intracellular Role in the Cytosolic Population.

The Salmonella invasion-associated type III secretion system (T3SS1) is an essential virulence factor required for entry into nonphagocytic cells and consequent uptake into a Salmonella-containing vacuole (SCV). While Salmonella is typically regarded as a vacuolar pathogen, a subset of bacteria escape from the SCV in epithelial cells and eventually hyperreplicate in the cytosol. T3SS1 is downregulated following bacterial entry into mammalian cells, but cytosolic Salmonella cells are T3SS1 induced, suggesting prolonged or resurgent activity of T3SS1 in this population. In order to investigate the postinternalization contributions of T3SS1 to the Salmonella infectious cycle in epithelial cells, we bypassed its requirement for bacterial entry by tagging the T3SS1-energizing ATPase InvC at the C terminus with peptides that are recognized by bacterial tail-specific proteases. This caused a dramatic increase in InvC turnover which rendered even assembled injectisomes inactive. Bacterial strains conditionally expressing these unstable InvC variants were proficient for invasion but underwent rapid and sustained intracellular inactivation of T3SS1 activity when InvC expression ceased. This allowed us to directly implicate T3SS1 activity in cytosolic colonization and bacterial egress. We subsequently identified two T3SS1-delivered effectors, SopB and SipA, that are required for efficient colonization of the epithelial cell cytosol. Overall, our findings support a multifaceted, postinvasion role for T3SS1 and its effectors in defining the cytosolic population of intracellular SalmonellaIMPORTANCE A needle-like apparatus, the type III secretion system (T3SS) injectisome, is absolutely required for Salmonella enterica to enter epithelial cells; this requirement has hampered the analysis of its postentry contributions. To identify T3SS1-dependent intracellular activities, in this study we overcame this limitation by developing a conditional inactivation in the T3SS whereby T3SS activity is chemically induced during culture in liquid broth, permitting bacterial entry into epithelial cells, but is quickly and perpetually inactivated in the absence of inducer. In this sense, the mutant acts like wild-type bacteria when extracellular and as a T3SS mutant once it enters a host cell. This "conditional" mutant allowed us to directly link activity of this T3SS with nascent vacuole lysis, cytosolic proliferation, and cellular egress, demonstrating that the invasion-associated T3SS also contributes to essential intracellular stages of the S. enterica infectious cycle.