Salmonella exploits membrane reservoirs for invasion of host cells.

Salmonella utilizes a type 3 secretion system to translocate virulence proteins (effectors) into host cells during infection1. The effectors modulate host cell machinery to drive uptake of the bacteria into vacuoles, where they can establish an intracellular replicative niche. A remarkable feature of Salmonella invasion is the formation of actin-rich protuberances (ruffles) on the host cell surface that contribute to bacterial uptake. However, the membrane source for ruffle formation and how these bacteria regulate membrane mobilization within host cells remains unclear. Here, we show that Salmonella exploits membrane reservoirs for the generation of invasion ruffles. The reservoirs are pre-existing tubular compartments associated with the plasma membrane (PM) and are formed through the activity of RAB10 GTPase. Under normal growth conditions, membrane reservoirs contribute to PM homeostasis and are preloaded with the exocyst subunit EXOC2. During Salmonella invasion, the bacterial effectors SipC, SopE2, and SopB recruit exocyst subunits from membrane reservoirs and other cellular compartments, thereby allowing exocyst complex assembly and membrane delivery required for bacterial uptake. Our findings reveal an important role for RAB10 in the establishment of membrane reservoirs and the mechanisms by which Salmonella can exploit these compartments during host cell invasion.

Kinase-independent synthesis of 3-phosphorylated phosphoinositides by a phosphotransferase.

Despite their low abundance, phosphoinositides play a central role in membrane traffic and signalling. PtdIns(3,4,5)P3 and PtdIns(3,4)P2 are uniquely important, as they promote cell growth, survival and migration. Pathogenic organisms have developed means to subvert phosphoinositide metabolism to promote successful infection and their survival in host organisms. We demonstrate that PtdIns(3,4)P2 is a major product generated in host cells by the effectors of the enteropathogenic bacteria Salmonella and Shigella. Pharmacological, gene silencing and heterologous expression experiments revealed that, remarkably, the biosynthesis of PtdIns(3,4)P2 occurs independently of phosphoinositide 3-kinases. Instead, we found that the Salmonella effector SopB, heretofore believed to be a phosphatase, generates PtdIns(3,4)P2 de novo via a phosphotransferase/phosphoisomerase mechanism. Recombinant SopB is capable of generating PtdIns(3,4,5)P3 and PtdIns(3,4)P2 from PtdIns(4,5)P2 in a cell-free system. Through a remarkable instance of convergent evolution, bacterial effectors acquired the ability to synthesize 3-phosphorylated phosphoinositides by an ATP- and kinase-independent mechanism, thereby subverting host signalling to gain entry and even provoke oncogenic transformation.

Salmonella effector SopD promotes plasma membrane scission by inhibiting Rab10.

Salmonella utilizes translocated virulence proteins (termed effectors) to promote host cell invasion. The effector SopD contributes to invasion by promoting scission of the plasma membrane, generating Salmonella-containing vacuoles. SopD is expressed in all Salmonella lineages and plays important roles in animal models of infection, but its host cell targets are unknown. Here we show that SopD can bind to and inhibit the small GTPase Rab10, through a C-terminal GTPase activating protein (GAP) domain. During infection, Rab10 and its effectors MICAL-L1 and EHBP1 are recruited to invasion sites. By inhibiting Rab10, SopD promotes removal of Rab10 and recruitment of Dynamin-2 to drive scission of the plasma membrane. Together, our study uncovers an important role for Rab10 in regulating plasma membrane scission and identifies the mechanism used by a bacterial pathogen to manipulate this function during infection.

Knockdown of genes involved in axonal transport enhances the toxicity of human neuromuscular disease-linked MATR3 mutations in Drosophila.

Mutations in the nuclear matrix protein Matrin 3 (MATR3) have been identified in amyotrophic lateral sclerosis and myopathy. To investigate the mechanisms underlying MATR3 mutations in neuromuscular diseases and efficiently screen for modifiers of MATR3 toxicity, we generated transgenic MATR3 flies. Our findings indicate that expression of wild-type or mutant MATR3 in motor neurons reduces climbing ability and lifespan of flies, while their expression in indirect flight muscles (IFM) results in abnormal wing positioning and muscle degeneration. In both motor neurons and IFM, mutant MATR3 expression results in more severe phenotypes than wild-type MATR3, demonstrating that the disease-linked mutations confer pathogenicity. We conducted a targeted candidate screen for modifiers of the MATR3 abnormal wing phenotype and identified multiple enhancers involved in axonal transport. Knockdown of these genes enhanced protein levels and insolubility of mutant MATR3. These results suggest that accumulation of mutant MATR3 contributes to toxicity and implicate axonal transport dysfunction in disease pathogenesis.

Intravoxel Incoherent Motion Combined With Dynamic Contrast-Enhanced Perfusion MRI of Early Cervical Carcinoma: Correlations Between Multimodal Parameters and HIF-1α Expression.

BACKGROUND: The identification of hypoxia inducible factor (HIF-1α) expression is helpful for the quantitative assessment of tumor hypoxia. The application of multimodal imaging techniques may play a part in the assessment of HIF-1α expression of cervical carcinoma. PURPOSE: To investigate the correlations between multiple imaging parameters and HIF-1α expression of early cervical carcinoma and to determine whether tumor hypoxia can be predicted using multisequence imaging parameters. STUDY TYPE: Prospective observational. POPULATION: One hundred patients with early cervical carcinoma. FIELD STRENGTH/SEQUENCES: 3.0 T MRI including intravoxel incoherent motion (IVIM) diffusion-weighted imaging (DWI) and dynamic contrast-enhanced (DCE) perfusion MRI sequences. ASSESSMENT: DCE-MRI and IVIM DWI were performed for all patients. The imaging parameters included volume transfer constant (Ktrans ), rate constant (Kep ), extravascular extracellular volume fraction (Ve ), D, D*, and f. STATISTICAL TESTS: The comparisons of imaging parameters between two independent groups were performed using the Mann-Whitney U-test. Multiple linear regression analysis was performed to determine the correlation between multiple imaging parameters and HIF-1α expression. The diagnostic ability of DCE-MRI, IVIM DWI, and the combination of two techniques for discriminating high-expression and low-expression groups were analyzed. RESULTS: The high-expression group had a lower Ktrans or Kep value than the low-expression group (P = 0.03; 0.02), while the high-expression group had a higher Ve value than the low-expression group (P = 0.03). The high-expression group had a higher D or f value than the low-expression group (P = 0.02; 0.02). Ktrans , Kep , D, Ve , and f values were independently correlated with HIF-1α expression. The sensitivity or accuracy of a combined method was higher than that of DCE-MRI or IVIM DWI individually (P = 0.03, 0.02; 0.04, 0.03). DATA CONCLUSION: The combination of DCE-MRI and IVIM DWI can improve the diagnostic ability of discriminating different HIF-1α expression levels for early cervical tumors. LEVEL OF EVIDENCE: 1 Technical Efficacy Stage: 2 J. Magn. Reson. Imaging 2019;50:918-929.

Intravoxel incoherent motion MR imaging of early cervical carcinoma: correlation between imaging parameters and tumor-stroma ratio.

OBJECTIVES: To investigate if intravoxel incoherent motion (IVIM) MR imaging can predict the tumour-stroma ratio (TSR) in patients with early cervical carcinoma. METHODS: Fifty-four patients with early cervical carcinoma were prospectively enrolled into this study. All patients underwent IVIM imaging and parameters including D, D* and f value were measured. The tumours were classified into stroma-rich and stroma-poor group according to TSR, and comparisons of IVIM parameters between two groups were performed. The relationships between IVIM parameters and TSR were analysed by using a multivariate multi-regression analysis. RESULTS: D and f values were significantly lower in stroma-poor tumours than in stroma-rich tumours (p=0.02, 0.04), while the difference in D* value between two groups didn't achieve statistical significance (p=0.09). The areas under ROC curves of D and f values in discriminating stroma-rich and stroma-poor tumours were 0.835 (95%CI=0.616~0.905) and 0.686 (95%CI=0.575~0.798). In multiple linear regression analysis, D value, pathologic type, histologic grade, tumour size and f value were independently correlated with TSR of cervical carcinoma. CONCLUSIONS: D and f values are independently correlated with TSR of cervical carcinoma and have the potential for quantitative measurement of TSR. KEY POINTS: • TSR is a recognized independent prognostic factor in many solid tumours. • D and f values measured by IVIM MRI are independently correlated with TSR while D* is not. • IVIM offers the potential to predict TSR.

The stratification of severity of acute radiation proctopathy after radiotherapy for cervical carcinoma using diffusion-weighted MRI.

OBJECTIVE: To determine whether diffusion-weighted imaging (DWI) can be used for quantitatively evaluating severity of acute radiation proctopathy after radiotherapy for cervical carcinoma. MATERIALS AND METHODS: One hundred and twenty-four patients with cervical carcinoma underwent MR examination including DWI before and after radiotherapy. Acute radiation proctopathy was classified into three groups (grade 0, grade I-II and grade III-IV) according to Toxicity Criteria of the Radiation Therapy Oncology Group (RTOG). The pretreatment ADC (ADCpre), ADC after treatment (ADCpost) and ADC change (ΔADC) were compared among three groups. In addition, acute radiation proctopathy was classified into good-prognosis group and poor-prognosis group. ADCpre, ADCpost and ΔADC were compared between two groups. For DWI parameter that had significant difference, discriminatory capability of the parameter was determined using receiver operating characteristics (ROC) analysis. RESULTS: ADCpost and ΔADC were higher in grade I-II group than in grade 0 group (p<0.05), yielding a sensitivity of 79.3% and specificity of 69.4% for ADCpost, and 85.1%, 72.3% for ΔADC for discrimination between two groups. ADCpost and ΔADC were higher in grade III-IV group than in grade I-II group (p<0.05), yielding a sensitivity of 80.3% and specificity of 72.5% for ADCpost, and 84.1%, 74.5% for ΔADC for discrimination between two groups. ADCpost and ΔADC were higher in poor-prognosis group than in good-prognosis group (p<0.05), yielding a sensitivity of 79.5% and specificity of 73.4% for ADCpost, and 87.2%, 78.3% for ΔADC for discrimination between two groups. CONCLUSION: Diffusion-weighted MRI can be used for quantitative stratification of severity of acute radiation proctopathy, which serves as an important basis for appropriate timely adjustment of radiotherapy for cervical carcinoma in order to maximally reduce the radiation injury of rectum.

Pulmonary fungal infections after bone marrow transplantation: the value of high-resolution computed tomography in predicting their etiology.

BACKGROUND: The correct diagnosis of etiology of fungal infection after bone marrow transplantation is very important to the choice of antifungal drugs and a premise for improvement of therapeutic efficacy. This study aimed to compare high-resolution computed tomography (HRCT) findings of the pulmonary fungal infections to determine whether the etiology of various fungal infections could be diagnosed with HRCT. METHODS: Eighty-five cases were enrolled. According to the pathogens responsible for fungal infections, the patients were classified into three groups including invasive aspergillosis (n = 52), candidiasis (n = 19) and cryptococcosis (n = 14) groups. All the patients underwent HRCT scans. Two independent radiologists retrospectively analyzed the HRCT scans regarding CT patterns and distribution of lung abnormality. RESULTS: Most fungal infections in the three groups occurred in the neutropenic phase. There was no significant difference in the constituent ratio of fungal infections at different phases after bone marrow transplantation among the three groups. Agreement between the two observers for all the CT characteristics of fungal infections was excellent (k > 0.75). There was a significant difference in occurrence ratio of mass among the three groups (P = 0.02). Occurrence ratio of mass (43.3%, 13/30) in the group with invasive aspergillosis was higher than in each of other two groups (20.0%, 2/10; 14.3%, 1/7). There was no significant difference in other CT characteristics of nodules or masses; including number, margin, halo sign, cavitation and air-crescent sign. There was no significant difference in number, margin, air bronchogram and distribution of air-space consolidation. CONCLUSIONS: The HRCT appearance of various pulmonary fungal infections has a great deal of overlap and is nonspecific. Mass is more common in invasive aspergillosis, which is helpful to the diagnosis of invasive aspergillosis after bone marrow transplantation.