Neisseria meningitidis Type IV Pili Composed of Sequence Invariable Pilins Are Masked by Multisite Glycosylation.

The ability of pathogens to cause disease depends on their aptitude to escape the immune system. Type IV pili are extracellular filamentous virulence factors composed of pilin monomers and frequently expressed by bacterial pathogens. As such they are major targets for the host immune system. In the human pathogen Neisseria meningitidis, strains expressing class I pilins contain a genetic recombination system that promotes variation of the pilin sequence and is thought to aid immune escape. However, numerous hypervirulent clinical isolates express class II pilins that lack this property. This raises the question of how they evade immunity targeting type IV pili. As glycosylation is a possible source of antigenic variation it was investigated using top-down mass spectrometry to provide the highest molecular precision on the modified proteins. Unlike class I pilins that carry a single glycan, we found that class II pilins display up to 5 glycosylation sites per monomer on the pilus surface. Swapping of pilin class and genetic background shows that the pilin primary structure determines multisite glycosylation while the genetic background determines the nature of the glycans. Absence of glycosylation in class II pilins affects pilus biogenesis or enhances pilus-dependent aggregation in a strain specific fashion highlighting the extensive functional impact of multisite glycosylation. Finally, molecular modeling shows that glycans cover the surface of class II pilins and strongly decrease antibody access to the polypeptide chain. This strongly supports a model where strains expressing class II pilins evade the immune system by changing their sugar structure rather than pilin primary structure. Overall these results show that sequence invariable class II pilins are cloaked in glycans with extensive functional and immunological consequences.

Early sequence of events triggered by the interaction of Neisseria meningitidis with endothelial cells.

Neisseria meningitidis is a bacterium responsible for severe sepsis and meningitis. Following type IV pilus-mediated adhesion to endothelial cells, bacteria proliferating on the cellular surface trigger a potent cellular response that enhances the ability of adhering bacteria to resist the mechanical forces generated by the blood flow. This response is characterized by the formation of numerous 100 nm wide membrane protrusions morphologically related to filopodia. Here, a high-resolution quantitative live-cell fluorescence microscopy procedure was designed and used to study this process. A farnesylated plasma membrane marker was first detected only a few seconds after bacterial contact, rapidly followed by actin cytoskeleton reorganization and bulk cytoplasm accumulation. The bacterial type IV pili-associated minor pilin PilV is necessary for the initiation of this cascade. Plasma membrane composition is a key factor as cholesterol depletion with methyl-ß-cyclodextrin completely blocks the initiation of the cellular response. In contrast membrane deformation does not require the actin cytoskeleton. Strikingly, plasma membrane remodelling undermicrocolonies is also independent of common intracellular signalling pathways as cellular ATP depletion is not inhibitory. This study shows that bacteria-induced plasma membrane reorganization is a rapid event driven by a direct cross-talk between type IV pili and the plasma membrane rather than by the activation of an intracellular signalling pathway that would lead to actin remodelling.

Complete posttranslational modification mapping of pathogenic Neisseria meningitidis pilins requires top-down mass spectrometry.

In pathogenic bacteria, posttranslationally modified proteins have been found to promote bacterial survival, replication, and evasion from the host immune system. In the human pathogen Neisseria meningitidis, the protein PilE (15-18 kDa) is the major building block of type IV pili, extracellular filamentous organelles that play a major role in mediating pathogenesis. Previous reports have shown that PilE can be expressed as a number of different proteoforms, each harboring its own set of PTMs and that specific proteoforms are key in promoting bacterial virulence. Efficient tools that allow complete PTM mapping of proteins involved in bacterial infection are therefore strongly needed. As we show in this study, a simple combination of mass profiling and bottom-up proteomics is fundamentally unable to achieve this goal when more than two proteoforms are present simultaneously. In a N. meningitidis strain isolated from a patient with meningitis, mass profiling revealed the presence of four major proteoforms of PilE, in a 1:1:1:1 ratio. Due to the complexity of the sample, a top-down approach was required to achieve complete PTM mapping for all four proteoforms, highlighting an unprecedented extent of glycosylation. Top-down MS therefore appears to be a promising tool for the analysis of highly posttranslationally modified proteins involved in bacterial virulence.

The tetraspanin CD9 controls migration and proliferation of parietal epithelial cells and glomerular disease progression.

The mechanisms driving the development of extracapillary lesions in focal segmental glomerulosclerosis (FSGS) and crescentic glomerulonephritis (CGN) remain poorly understood. A key question is how parietal epithelial cells (PECs) invade glomerular capillaries, thereby promoting injury and kidney failure. Here we show that expression of the tetraspanin CD9 increases markedly in PECs in mouse models of CGN and FSGS, and in kidneys from individuals diagnosed with these diseases. Cd9 gene targeting in PECs prevents glomerular damage in CGN and FSGS mouse models. Mechanistically, CD9 deficiency prevents the oriented migration of PECs into the glomerular tuft and their acquisition of CD44 and ß1 integrin expression. These findings highlight a critical role for de novo expression of CD9 as a common pathogenic switch driving the PEC phenotype in CGN and FSGS, while offering a potential therapeutic avenue to treat these conditions.

Adhesion to nanofibers drives cell membrane remodeling through one-dimensional wetting.

The shape of cellular membranes is highly regulated by a set of conserved mechanisms that can be manipulated by bacterial pathogens to infect cells. Remodeling of the plasma membrane of endothelial cells by the bacterium Neisseria meningitidis is thought to be essential during the blood phase of meningococcal infection, but the underlying mechanisms are unclear. Here we show that plasma membrane remodeling occurs independently of F-actin, along meningococcal type IV pili fibers, by a physical mechanism that we term 'one-dimensional' membrane wetting. We provide a theoretical model that describes the physical basis of one-dimensional wetting and show that this mechanism occurs in model membranes interacting with nanofibers, and in human cells interacting with extracellular matrix meshworks. We propose one-dimensional wetting as a new general principle driving the interaction of cells with their environment at the nanoscale that is diverted by meningococci during infection.

Loss of HIF-1α in natural killer cells inhibits tumour growth by stimulating non-productive angiogenesis.

Productive angiogenesis, a prerequisite for tumour growth, depends on the balanced release of angiogenic and angiostatic factors by different cell types within hypoxic tumours. Natural killer (NK) cells kill cancer cells and infiltrate hypoxic tumour areas. Cellular adaptation to low oxygen is mediated by Hypoxia-inducible factors (HIFs). We found that deletion of HIF-1α in NK cells inhibited tumour growth despite impaired tumour cell killing. Tumours developing in these conditions were characterised by a high-density network of immature vessels, severe haemorrhage, increased hypoxia, and facilitated metastasis due to non-productive angiogenesis. Loss of HIF-1α in NK cells increased the bioavailability of the major angiogenic cytokine vascular endothelial growth factor (VEGF) by decreasing the infiltration of NK cells that express angiostatic soluble VEGFR-1. In summary, this identifies the hypoxic response in NK cells as an inhibitor of VEGF-driven angiogenesis, yet, this promotes tumour growth by allowing the formation of functionally improved vessels.

TLX homeodomain oncogenes mediate T cell maturation arrest in T-ALL via interaction with ETS1 and suppression of TCRα gene expression.

Acute lymphoblastic leukemias (ALLs) are characterized by multistep oncogenic processes leading to cell-differentiation arrest and proliferation. Specific abrogation of maturation blockage constitutes a promising therapeutic option in cancer, which requires precise understanding of the underlying molecular mechanisms. We show that the cortical thymic maturation arrest in T-lineage ALLs that overexpress TLX1 or TLX3 is due to binding of TLX1/TLX3 to ETS1, leading to repression of T cell receptor (TCR) α enhanceosome activity and blocked TCR-Jα rearrangement. TLX1/TLX3 abrogation or enforced TCRαß expression leads to TCRα rearrangement and apoptosis. Importantly, the autoextinction of clones carrying TCRα-driven TLX1 expression supports TLX "addiction" in TLX-positive leukemias and provides further rationale for targeted therapy based on disruption of TLX1/TLX3.

Prognostic and oncogenic relevance of TLX1/HOX11 expression level in T-ALLs.

TLX1 is a homeodomain transcription factor generally associated with a favorable outcome in T-cell acute lymphoblastic leukemia (T-ALL). However, the molecular mechanisms of TLX1 deregulation remain unclear and various transcript levels in the absence of 10q24 abnormalities have been reported. A reproducible and accurate delineation of TLX1(+) T-ALL will be necessary for proper therapeutic stratification. We have studied 264 unselected T-ALLs (171 adults and 93 children) and show that T-ALLs expressing high levels of TLX1 (n = 35, 13%), defined as a real-time quantitative polymerase chain reaction (RQ-PCR) level of TLX1 greater than 1.00 ABL, form a homogeneous oncogenic group, based on their uniform stage of maturation arrest and oncogenetic and transcriptional profiles. Furthermore, TLX1-high T-ALLs harbor molecular TLX1 locus abnormalities in the majority (31/33), a proportion largely underestimated by standard karyotypic screening. T-ALLs expressing TLX1 at lower levels (n = 57, 22%) do not share these characteristics. Prognostic analysis within the adult LALA94 and GRAALL03 prospective protocols demonstrate a better event-free survival (P = .035) and a marked trend for longer overall survival (P = .059) for TLX1-high T-ALLs, while the expression of lower levels of TLX1 does not impact on prognosis. We propose that TLX1(+) T-ALLs be defined as cases expressing TLX1/ABL ratios greater than 1 and/or demonstrating TLX1 rearrangement. Therapeutic modification should be considered for those patients.

Expression of T-lineage-affiliated transcripts and TCR rearrangements in acute promyelocytic leukemia: implications for the cellular target of t(15;17).

Acute promyelocytic leukemia (APL) is the most differentiated form of acute myeloid leukemia (AML) and has generally been considered to result from transformation of a committed myeloid progenitor. Paradoxically, APL has long been known to express the T-cell lymphoid marker, CD2. We searched for other parameters indicative of T-cell lymphoid specification in a cohort of 36 APL cases, revealing a frequent but asynchronous T-cell lymphoid program most marked in the hypogranular variant (M3v) subtype, with expression of PTCRA, sterile TCRA, and TCRG transcripts and TCRG rearrangement in association with sporadic cytoplasmic expression of CD3 or TdT proteins. Gene-expression profiling identified differentially expressed transcription factors that have been implicated in lymphopoiesis. These data carry implications for the hematopoietic progenitor targeted by the PML-RARA oncoprotein in APL and are suggestive of a different cellular origin for classic hypergranular (M3) and variant forms of the disease. They are also consistent with the existence and subsequent transformation of progenitor populations with lymphoid/myeloid potential.