Deep mutational scanning of the Neisseria meningitidis major pilin reveals the importance of pilus tip-mediated adhesion.

Type IV pili (TFP) are multifunctional micrometer-long filaments expressed at the surface of many prokaryotes. In Neisseria meningitidis, TFP are crucial for virulence. Indeed, these homopolymers of the major pilin PilE mediate interbacterial aggregation and adhesion to host cells. However, the mechanisms behind these functions remain unclear. Here, we simultaneously determined regions of PilE involved in pilus display, auto-aggregation, and adhesion by using deep mutational scanning and started mining this extensive functional map. For auto-aggregation, pili must reach a minimum length to allow pilus-pilus interactions through an electropositive cluster of residues centered around Lys140. For adhesion, results point to a key role for the tip of the pilus. Accordingly, purified pili interacting with host cells initially bind via their tip-located major pilin and then along their length. Overall, these results identify functional domains of PilE and support a direct role of the major pilin in TFP-dependent aggregation and adhesion.

Adaptation of commensal proliferating Escherichia coli to the intestinal tract of young children with cystic fibrosis.

The mature human gut microbiota is established during the first years of life, and altered intestinal microbiomes have been associated with several human health disorders. Escherichia coli usually represents less than 1% of the human intestinal microbiome, whereas in cystic fibrosis (CF), greater than 50% relative abundance is common and correlates with intestinal inflammation and fecal fat malabsorption. Despite the proliferation of E. coli and other Proteobacteria in conditions involving chronic gastrointestinal tract inflammation, little is known about adaptation of specific characteristics associated with microbiota clonal expansion. We show that E. coli isolated from fecal samples of young children with CF has adapted to growth on glycerol, a major component of fecal fat. E. coli isolates from different CF patients demonstrate an increased growth rate in the presence of glycerol compared with E. coli from healthy controls, and unrelated CF E. coli strains have independently acquired this growth trait. Furthermore, CF and control E. coli isolates have differential gene expression when grown in minimal media with glycerol as the sole carbon source. While CF isolates display a growth-promoting transcriptional profile, control isolates engage stress and stationary-phase programs, which likely results in slower growth rates. Our results indicate that there is selection of unique characteristics within the microbiome of individuals with CF, which could contribute to individual disease outcomes.

AquaSparkTM - Rapid Environmental Monitoring of Listeria monocytogenes.

In order to prevent microbial contamination of food, monitoring of the production environment, together with the rapid detection of foodborne pathogens have proven to be of utmost importance for Food Safety. Environmental monitoring should detect harmful pathogens at the earliest point in time in order for the necessary interventions to be taken. However, current detection methods fall short with regards to speed, ease of use, and cost. This article aims to present the idea behind NEMIS Technologies, a startup company making use of the novel AquaSparkTM technology for the development of a new generation of bacterial detection methods. These methods utilize chemiluminescence in order to detect live target bacteria in a short period of time compared to that of conventional methods. We show that dry-stressed Listeria monocytogenes can be detected within 24 hours, using small-molecule chemiluminescent probes, together with a bacteria-specific proprietary enrichment broth containing a cocktail of bacteriophages, which enhance the specificity and sensitivity. This novel platform technology has the potential to extend beyond environmental monitoring towards food analyses as well as veterinary and human health.

The number of Neisseria meningitidis type IV pili determines host cell interaction.

As mediators of adhesion, autoaggregation and bacteria-induced plasma membrane reorganization, type IV pili are at the heart of Neisseria meningitidis infection. Previous studies have proposed that two minor pilins, PilV and PilX, are displayed along the pilus structure and play a direct role in mediating these effects. In contrast with this hypothesis, combining imaging and biochemical approaches we found that PilV and PilX are located in the bacterial periplasm rather than along pilus fibers. Furthermore, preventing exit of these proteins from the periplasm by fusing them to the mCherry protein did not alter their function. Deletion of the pilV and pilX genes led to a decrease in the number, but not length, of pili displayed on the bacterial surface indicating a role in the initiation of pilus biogenesis. By finely regulating the expression of a central component of the piliation machinery, we show that the modest reductions in the number of pili are sufficient to recapitulate the phenotypes of the pilV and pilX mutants. We further show that specific type IV pili-dependent functions require different ranges of pili numbers.

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.

Validation of N-Light™ Salmonella Risk Test Kit for Detection of Salmonella spp. on Environmental Surfaces: AOAC Performance Tested MethodSM 072204.

BACKGROUND: The NEMIS N-Light™ Salmonella Risk method uses chemiluminescence designed for the qualitative detection of Salmonella spp. from environmental surface samples. OBJECTIVE: To validate the N-Light Salmonella Risk assay using independent and method developer validation studies according to the AOAC Performance Tested MethodsSM (PTM) program for the detection of Salmonella spp. on stainless-steel, polystyrene, and ceramic environmental surfaces. METHOD: The N-Light Salmonella Risk assay was evaluated in a matrix study in comparison to the ISO 6579-1:2017 method ("Microbiology of the Food Chain-Horizontal Method for the Detection, Enumeration, and Serotyping of Salmonella-Part 1: Detection of Salmonella spp.") using an unpaired study design. Additional PTM studies performed were inclusivity/exclusivity, robustness, product consistency, and stability. RESULTS: The N-Light Salmonella Risk assay demonstrated a specific detection of all Salmonella strains tested. In the matrix study, the N-Light Salmonella Risk assay showed no significant differences between presumptive and confirmed results or between candidate and reference method results on the three surfaces evaluated. Data for additional PTM studies met acceptance criteria requirements. CONCLUSIONS: The NEMIS Technologies N-Light Salmonella Risk assay is an effective method for the qualitative detection of Salmonella on stainless-steel, polystyrene, and ceramic environmental surfaces. HIGHLIGHTS: The NEMIS Technologies N-Light Salmonella Risk assay, which is the first chemiluminescence-based detection system that uses a novel, patented dioxetane compound, allowing for easy and rapid detection of Salmonella.

Posttranslational modification of pili upon cell contact triggers N. meningitidis dissemination.

The Gram-negative bacterium Neisseria meningitidis asymptomatically colonizes the throat of 10 to 30% of the human population, but throat colonization can also act as the port of entry to the blood (septicemia) and then the brain (meningitis). Colonization is mediated by filamentous organelles referred to as type IV pili, which allow the formation of bacterial aggregates associated with host cells. We found that proliferation of N. meningitidis in contact with host cells increased the transcription of a bacterial gene encoding a transferase that adds phosphoglycerol onto type IV pili. This unusual posttranslational modification specifically released type IV pili-dependent contacts between bacteria. In turn, this regulated detachment process allowed propagation of the bacterium to new colonization sites and also migration across the epithelium, a prerequisite for dissemination and invasive disease.