Transporters MRP1 and MRP2 Regulate Opposing Inflammatory Signals To Control Transepithelial Neutrophil Migration during Streptococcus pneumoniae Lung Infection.

Streptococcus pneumoniae remains a source of morbidity and mortality in both developed and underdeveloped nations of the world. Disease can manifest as pneumonia, bacteremia, and meningitis, depending on the localization of infection. Interestingly, there is a correlation in experimental murine infections between the development of bacteremia and influx of neutrophils into the pulmonary lumen. Reduction of this neutrophil influx has been shown to improve survivability during infection. In this study, we use in vitro biotinylation and neutrophil transmigration and in vivo murine infection to identify a system in which two epithelium-localized ATP-binding cassette transporters, MRP1 and MRP2, have inverse activities dictating neutrophil transmigration into the lumen of infected mouse lungs. MRP1 effluxes an anti-inflammatory molecule that maintains homeostasis in uninfected contexts, thus reducing neutrophil infiltration. During inflammatory events, however, MRP1 decreases and MRP2 both increases and effluxes the proinflammatory eicosanoid hepoxilin A3. If we then decrease MRP2 activity during experimental murine infection with S. pneumoniae, we reduce both neutrophil infiltration and bacteremia, showing that MRP2 coordinates this activity in the lung. We conclude that MRP1 assists in depression of polymorphonuclear cell (PMN) migration by effluxing a molecule that inhibits the proinflammatory effects of MRP2 activity.IMPORTANCEStreptococcus pneumoniae is a Gram-positive bacterium that normally inhabits the human nasopharynx asymptomatically. However, it is also a major cause of pneumonia, bacteremia, and meningitis. The transition from pneumonia to bacteremia is critical, as patients that develop septicemia have ~20% mortality rates. Previous studies have shown that while neutrophils, a major bacterium-induced leukocyte, aid in S. pneumoniae elimination, they also contribute to pathology and may mediate the lung-to-blood passage of the bacteria. Herein, we show that epithelium-derived MRP1 and MRP2 efflux immunomodulatory agents that assist in controlling passage of neutrophils during infection and that limiting neutrophil infiltration produced less bacteremia and better survival during murine infection. The importance of our work is twofold: ours is the first to identify an MRP1/MRP2 axis of neutrophil control in the lung. The second is to provide possible therapeutic targets to reduce excess inflammation, thus reducing the chances of developing bacteremia during pneumococcal pneumonia.

A phosphorylated pseudokinase complex controls cell wall synthesis in mycobacteria.

Prokaryotic cell wall biosynthesis is coordinated with cell growth and division, but the mechanisms regulating this dynamic process remain obscure. Here, we describe a phosphorylation-dependent regulatory complex that controls peptidoglycan (PG) biosynthesis in Mycobacterium tuberculosis. We found that PknB, a PG-responsive Ser-Thr protein kinase (STPK), initiates complex assembly by phosphorylating a kinase-like domain in the essential PG biosynthetic protein, MviN. This domain was structurally diverged from active kinases and did not mediate phosphotransfer. Threonine phosphorylation of the pseudokinase domain recruited the FhaA protein through its forkhead-associated (FHA) domain. The crystal structure of this phosphorylated pseudokinase-FHA domain complex revealed the basis of FHA domain recognition, which included unexpected contacts distal to the phosphorylated threonine. Conditional degradation of these proteins in mycobacteria demonstrated that MviN was essential for growth and PG biosynthesis and that FhaA regulated these processes at the cell poles and septum. Controlling this spatially localized PG regulatory complex is only one of several cellular roles ascribed to PknB, suggesting that the capacity to coordinate signaling across multiple processes is an important feature conserved between eukaryotic and prokaryotic STPK networks.

TM4SF1: a tetraspanin-like protein necessary for nanopodia formation and endothelial cell migration.

Transmembrane-4-L-six-family-1 (TM4SF1) is a tetraspanin-like membrane protein that is highly and selectively expressed by cultured endothelial cells (EC) and, in vivo, by EC lining angiogenic tumor blood vessels. TM4SF1 is necessary for the formation of unusually long (up to a 50 µm), thin (~100-300 nm wide), F-actin-poor EC cell projections that we term 'nanopodia'. Immunostaining of nanopodia at both the light and electron microsopic levels localized TM4SF1 in a regularly spaced, banded pattern, forming TM4FS1-enriched domains. Live cell imaging of GFP-transduced HUVEC demonstrated that EC project nanopodia as they migrate and interact with neighboring cells. When TM4SF1 mRNA levels in EC were increased from the normal ~90 mRNA copies/cell to ~400 copies/cell through adenoviral transduction, EC projected more and longer nanopodia from the entire cell circumference but were unable to polarize or migrate effectively. When fibroblasts, which normally express TM4SF1 at ~5 copies/cell, were transduced to express TM4SF1 at EC-like levels, they formed typical TM4SF1-banded nanopodia, and broadened, EC-like lamellipodia. Mass-spectrometry demonstrated that TM4SF1 interacted with myosin-10 and ß-actin, proteins involved in filopodia formation and cell migration. In summary, TM4SF1, like genuine tetraspanins, serves as a molecular organizer that interacts with membrane and cytoskeleton-associated proteins and uniquely initiates the formation of nanopodia and facilitates cell polarization and migration.

A multi-gene transcriptional profiling approach to the discovery of cell signature markers.

A profile of transcript abundances from multiple genes constitutes a molecular signature if the expression pattern is unique to one cell type. Here we measure mRNA copy numbers per cell by normalizing per million copies of 18S rRNA and identify 6 genes (TIE1, KDR, CDH5, TIE2, EFNA1 and MYO5C) out of 79 genes tested as excellent molecular signature markers for endothelial cells (ECs) in vitro. The selected genes are uniformly expressed in ECs of 4 different origins but weakly or not expressed in 4 non-EC cell lines. A multi-gene transcriptional profile of these 6 genes clearly distinguishes ECs from non-ECs in vitro. We conclude that (i) a profile of mRNA copy numbers per cell from a well-chosen multi-gene panel can act as a sensitive and accurate cell type signature marker, and (ii) the method described here can be applied to in vivo cell fingerprinting and molecular diagnosis.

The L6 protein TM4SF1 is critical for endothelial cell function and tumor angiogenesis.

Transmembrane-4-L-six-family-1 (TM4SF1) was originally described as a cancer cell protein. Here, we show that it is highly expressed in the vascular endothelium of human cancers and in a banded pattern in the filopodia of cultured endothelial cells (EC). TM4SF1 knockdown prevented filopodia formation, inhibited cell mobility, blocked cytokinesis, and rendered EC senescent. Integrin-alpha5 and integrin-beta1 subunits gave a similar staining pattern and interacted constitutively with TM4SF1, whereas integrin subunits often associated with angiogenesis (alphaV, beta3, beta5) interacted with TM4SF1 only after vascular endothelial growth factor (VEGF)-A or thrombin stimulation. TM4SF1 knockdown substantially inhibited maturation of VEGF-A(164)-induced angiogenesis. Thus, TM4SF1 is a key regulator of EC function in vitro and of pathologic angiogenesis in vivo and is potentially an attractive target for antiangiogenesis therapy.