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bioRxiv2018

Aspartyl Protease 5 matures virulence factors found at the host-parasite interface in Toxoplasma gondii

Toxoplasma gondii infects approximately 30% of the worlds population, causing disease primarily during pregnancy and in individuals with weakened immune systems. Toxoplasma secretes and exports effector proteins that modulate the host during infection and several of these proteins are processed by the Golgi-associated Aspartyl Protease 5 (ASP5). Here, we identify ASP5 substrates by selectively enriching N-terminally-derived peptides from wildtype and {Delta}asp5 parasites. We reveal over two thousand unique Toxoplasma N-terminal peptides, mapping to both natural N-termini and protease cleavage sites. Several of these peptides mapped directly downstream of the characterised ASP5-cleavage site, arginine-arginine-leucine (RRL). We validate candidates as true ASP5 substrates, revealing they are not processed in parasites lacking ASP5, nor in wild type parasites following mutation of the motif from RRL{longrightarrow}ARL. All new ASP5 substrates are dense granule proteins, and interestingly none appear to be exported, thus differing from the analogous system in related Plasmodium spp., instead revealing that the majority of substrates reside within the parasitophorous vacuole (PV), and its membrane (the PVM), including two kinases and one phosphatase. Furthermore, we show that several of these ASP5-substrates are virulence factors, with their removal leading to attenuation in a mouse model, suggesting that phosphorylation at the host-parasite interface is important for virulence. Collectively, these data constitute the first in-depth analyses of the total list of ASP5 substrates, and shed new light on the role of ASP5 as a maturase of dense granule proteins during the Toxoplasma lytic cycle.

microbiology↗

bioRxiv2018

Identification of changing ribosome protein compositions using cryo-EM and mass spectrometry

The regulatory role of the ribosome in gene expression has come into sharper focus. It has been proposed that ribosomes are dynamic complexes capable of changing their protein composition in response to enviromental stimuli. We applied both cryo-EM and mass spectrometry to identify such changes in S. cerevisiae 80S ribosomes. Cryo-EM shows a fraction (17%) of the ribosome population in yeast growing in glucose lack the ribosomal proteins RPL10 (ul16) and RPS1A/B (eS1). Unexpectedly, this fraction rapidly increases to 34% after the yeast are switched to growth in glycerol. Using quantitative mass spectrometry, we found that the paralog yeast ribosomal proteins RPL8A (eL8A) and RPL8B (eL8B) change their relative proportions in the 80S ribosome when yeast are switched from growth in glucose to glycerol. Using yeast genetics and polysome profiling, we show that yeast ribosomes containing either RPL8A or RPL8B are not functionally interchangeable. Our combined cryo-EM and quantitative proteomic data support the hypothesis that ribosomes are dynamic complexes that alter their composition and functional activity in response to changes in growth or environmental conditions.

microbiology↗

bioRxiv2018

Specific enrichment of hyperthermophilic electroactive Archaea from deep-sea hydrothermal vent on electrically conductive support

While more and more investigations are done to isolate hyperthermophilic exoelectrogenic communities from environments, none have been performed yet on deep-sea hydrothermal vent. Samples of black smoker chimney from Rainbow site on the Atlantic mid-oceanic ridge have been harvested for enriching exoelectrogens in microbial electrolysis cells under hyperthermophilic (80{degrees}C) condition. Two enrichments have been performed: one from direct inoculation of crushed chimney and the other one from inoculation of a pre-cultivation on iron (III) oxide. In both experiments, a current production was observed from 2.4 A/m2 to 5.8 A/m2 with a set anode potential of +0.05 vs SHE. Taxonomic affiliation of the exoelectrogen communities obtained exhibited a specific enrichment of Archaea from Thermococcales and Archeoglobales orders on the electrode, even when both inocula were dominated by Bacteria.

microbiology↗

bioRxiv2018

Improved sgRNA design in bacteria via genome-wide activity profiling

CRISPR/Cas9 is a promising tool in prokaryotic genome engineering, but its success is limited by the widely varying on-target activity of single guide RNAs (sgRNAs). Based on the association of CRISPR/Cas9-induced DNA cleavage with cellular lethality, we systematically profiled sgRNA activity by co-expressing a genome-scale library (~70,000 sgRNAs) with Cas9 or its specificity-improved mutant in E. coli. Based on this large-scale dataset, we constructed a comprehensive and high-density sgRNA activity map, which enables selecting highly active sgRNAs for any locus across the genome in this model organism. We also identified resistant genomic loci with respect to CRISPR/Cas9 activity, notwithstanding the highly accessible DNA in bacterial cells. Moreover, we found that previous sgRNA activity prediction models that were trained on mammalian cell datasets were inadequate when coping with our results, highlighting the key limitations and biases of previous models. We hence developed an integrated algorithm to accurately predict highly effective sgRNAs, aiming to facilitate the design of CRISPR/Cas9-based genome engineering or screenings in bacteria. We also isolated the important sgRNA features that contribute to DNA cleavage and characterized their key differences among wild type Cas9 and its mutant, shedding light on the biophysical mechanisms of the CRISPR/Cas9 system.

microbiology↗

bioRxiv2018

Group IIA secreted phospholipase A2 in human serum kills commensal but not clinical Enterococcus faecium isolates

Human innate immunity employs cellular and humoral mechanisms to facilitate rapid killing of invading bacteria. The direct killing of bacteria by human serum is mainly attributed to the activity of the complement system that forms pores in Gram-negative bacteria. Although Gram-positive bacteria are considered resistant to serum killing, we here uncover that normal human serum effectively kills Enterococcus faecium. Comparison of a well-characterized collection of commensal and clinical E. faecium isolates revealed that human serum specifically kills commensal E. faecium strains isolated from normal gut microbiota, but not clinical isolates. Inhibitor studies show that the human group IIA secreted phospholipase A2 (hGIIA), but not complement, is responsible for killing of commensal E. faecium strains in human normal serum. This is remarkable since hGIIA concentrations in non-inflamed serum were considered too low to be bactericidal against Gram-positive bacteria. Mechanistic studies showed that serum hGIIA specifically causes permeabilization of commensal E. faecium membranes. Altogether, we find that a normal serum concentration of hGIIA effectively kills commensal E. faecium and that hGIIA resistance of clinical E. faecium could have contributed to the ability of these strains to become opportunistic pathogens in hospitalized patients.\n\nImportanceHuman normal serum contains antimicrobial components that effective kill invading Gram-negative bacteria. Although Gram-positive bacteria are generally considered resistant to serum killing, here we show that normal human effectively kills the Gram-positive Enterococcus faecium strains that live as commensals in the gut of humans. In contrast, clinical E. faecium strains that are responsible for opportunistic infections in debilitated patients are resistant against human serum. The key factor in serum responsible for killing is group IIA secreted phospholipase A2 (hGIIA) that effectively destabilizes commensal E. faecium membranes. We believe that hGIIA resistance by clinical E. faecium could have contributed to the ability of these strains to cause opportunistic infections in hospitalized patients. Altogether, understanding mechanisms of immune defense and bacterial resistance could aid in further development of novel anti-infective strategies against medically important multidrug resistant Gram-positive pathogens.

microbiology↗

bioRxiv2018

Live Zika virus chimeric vaccine candidate based on a yellow fever 17-D attenuated backbone

Zika virus (ZIKV) recently dispersed throughout the tropics and sub-tropics causing epidemics associated with congenital disease and neurological complications. There is currently no commercial vaccine for ZIKV. Here we describe the initial development of a chimeric virus containing the prM/E proteins of a ZIKV epidemic strain incorporated into a yellow fever 17-D attenuated backbone. Using the versatile and rapid ISA (Infectious Subgenomic Amplicons) reverse genetics method, we compared different constructs and confirmed the need to modify the cleavage site between the pre-peptide and prM protein. Genotypic characterization of the chimeras indicates that emergence of compensatory mutations in the E protein is required to restore virus replicative fitness. Using an immunocompromised mouse model, we demonstrate that mice infected with the chimeric virus produced levels of neutralizing antibodies close to those observed following infection with ZIKV and that pre-immunized mice were protected against viscerotropic and neuroinvasive virus following challenge with a heterologous strain of ZIKV. These data provide a sound basis for the future development of this ZIKV vaccine candidate.

microbiology↗

bioRxiv2018

A catalog of microbial genes from the bovine rumen reveals the determinants of herbivory

BackgroundThe rumen microbiota provides essential services to its host and, through its role in ruminant production, contributes to human nutrition and food security. A thorough knowledge of the genetic potential of rumen microbes will provide opportunities for improving the sustainability of ruminant production systems. The availability of gene reference catalogs from gut microbiomes has advanced the understanding of the role of the microbiota in health and disease in humans and other mammals. In this work, we established a catalog of reference prokaryote genes from the bovine rumen.\n\nResultsUsing deep metagenome sequencing we identified 13,825,880 non-redundant prokaryote genes from the bovine rumen. Compared to human, pig and mouse gut metagenome catalogs, the rumen is larger and richer in functions and microbial species associated with the degradation of plant cell wall material and production of methane. Genes encoding enzymes catalyzing the breakdown of plant polysaccharides showed a particularly high richness that is otherwise impossible to infer from available genomes or shallow metagenomics sequencing. The catalog expands by several folds the dataset of carbohydrate-degrading enzymes described in the rumen. Using an independent dataset from a group of 77 cattle fed 4 common dietary regimes, we found that only <0.1% of genes were shared by all animals, which contrast with a large overlap for functions, i.e. 63% for KEGG functions. Different diets induced differences in the relative abundance rather than the presence or absence of genes explaining the great adaptability of cattle to rapidly adjust to dietary changes.\n\nConclusionsThese data bring new insights into functions, carbohydrate-degrading enzymes and microbes of the rumen that is complementing the available information on microbial genomes. The catalog is a significant biological resource enabling deeper understanding of phenotypes and biological processes and will be expanded as new data is made available.

microbiology↗

bioRxiv2018

Genetic recombination of poliovirus facilitates subversion of host barriers to infection

The contribution of RNA recombination to viral fitness and pathogenesis is poorly defined. Here, we isolate a recombination-deficient, poliovirus variant and find that, while recombination is detrimental to virus replication in tissue culture, recombination is important for pathogenesis in infected animals. Notably, recombination-defective virus exhibits severe attenuation following intravenous inoculation that is associated with a significant reduction in population size during intra-host spread. Because the impact of high mutational loads manifests most strongly at small population sizes, our data suggest that the repair of mutagenized genomes is an essential function of recombination and that this function may drive the long-term maintenance of recombination in viral species despite its associated fitness costs.\n\nSignificance StatementRNA recombination is a widespread but poorly understood feature of RNA virus replication. For poliovirus, recombination is involved in the emergence of neurovirulent circulating vaccine-derived poliovirus, which has hampered global poliovirus eradication efforts. This emergence illustrates the power of recombination to drive major adaptive change; however, it remains unclear if these adaptive events represent the primary role of recombination in virus survival. Here, we identify a viral mutant with a reduced rate of recombination and find that recombination also plays a central role in the spread of virus within animal hosts. These results highlight a novel approach for improving the safety of live attenuated vaccines and further our understanding of the role of recombination in virus pathogenesis and evolution.

microbiology↗

bioRxiv2018

Performance of convolutional neural networks for identification of bacteria in 3D microscopy datasets

Three-dimensional microscopy is increasingly prevalent in biology due to the development of techniques such as multiphoton, spinning disk confocal, and light sheet fluorescence microscopies. These methods enable unprecedented studies of life at the microscale, but bring with them larger and more complex datasets. New image processing techniques are therefore called for to analyze the resulting images in an accurate and efficient manner. Convolutional neural networks are becoming the standard for classification of objects within images due to their accuracy and generalizability compared to traditional techniques. Their application to data derived from 3D imaging, however, is relatively new and has mostly been in areas of magnetic resonance imaging and computer tomography. It remains unclear, for images of discrete cells in variable backgrounds as are commonly encountered in fluorescence microscopy, whether convolutional neural networks provide sufficient performance to warrant their adoption, especially given the challenges of human comprehension of their classification criteria and their requirements of large training datasets. We therefore applied a 3D convolutional neural network to distinguish bacteria and non-bacterial objects in 3D light sheet fluorescence microscopy images of larval zebrafish intestines. We find that the neural network is as accurate as human experts, outperforms random forest and support vector machine classifiers, and generalizes well to a different bacterial species through the use of transfer learning. We also discuss network design considerations, and describe the dependence of accuracy on dataset size and data augmentation. We provide source code, labeled data, and descriptions of our analysis pipeline to facilitate adoption of convolutional neural network analysis for three-dimensional microscopy data.\n\nAuthor summaryThe abundance of complex, three dimensional image datasets in biology calls for new image processing techniques that are both accurate and fast. Deep learning techniques, in particular convolutional neural networks, have achieved unprecedented accuracies and speeds across a large variety of image classification tasks. However, it is unclear whether or not their use is warranted in noisy, heterogeneous 3D microscopy datasets, especially considering their requirements of large, labeled datasets and their lack of comprehensible features. To asses this, we provide a case study, applying convolutional neural networks as well as feature-based methods to light sheet fluorescence microscopy datasets of bacteria in the intestines of larval zebrafish. We find that the neural network is as accurate as human experts, outperforms the feature-based methods, and generalizes well to a different bacterial species through the use of transfer learning.

microbiology↗

bioRxiv2018

Humidity as a non-pharmaceutical intervention for influenza A

Influenza is a global problem infecting 5-10 % of adults and 20-30 % of children annually. Non-pharmaceutical interventions (NPIs) are attractive approaches to complement vaccination in the prevention and reduction of influenza. Strong cyclical reduction of absolute humidity has been associated with influenza outbreaks in temperate climates. This study tested the hypothesis that raising absolute humidity above seasonal lows would impact influenza virus survival and transmission in a key source of influenza distribution, a community school. Air samples and objects handled by students (e.g. blocks and markers) were collected from preschool classrooms. All samples were processed and PCR used to determine the presence of influenza and its amount. Additionally samples were tested for their ability to infect cells in cultures. Deliberate classroom humidification (with commercial steam humidifiers) resulted in a significant reduction of the total number of influenza positive samples (air and fomite), viral copy number, and efficiency of viral infectivity. This is the first prospective study suggesting that exogenous humidification could serve as a scalable NPI for influenza or other viral outbreaks.\n\nAuthor summaryHuman influenza infections have a substantial impact on society (including lost productivity and medical costs). Children, 3-4 years of age are the main introducers and spreaders of influenza within a household and community. There is evidence from laboratory and epidemiological studies that suggests that low humidity in winter (in temperate climates) may increase the ability of influenza virus to survive and spread between individuals. We wanted to know if added in humidity (through steam humidifiers) could reduce the amount of influenza present and its spread within preschool classrooms (students aged 3-4 years)? Additionally, we looked at the infectivity of the influenza isolated and if there were differences in the number of students with influenza-like illnesses during our study. We show that humidification can reduce the amount of influenza present within samples from preschool classrooms and that there were fewer infectious samples compared to non-humidified rooms. There were small numbers of students ill with influenza like illnesses during our study so additional studies will need to look further at humidification as a way to reduce influenza infection and transmission.

microbiology↗

bioRxiv2018

Comparative Analysis of UL16 Mutants Derived from Multiple Strains of HSV-2 and HSV-1 Reveals Species-Specific Requirements for the UL16 Protein

Orthologs of the herpes simplex virus (HSV) UL16 gene are conserved throughout the Herpesviridae. Because of this conservation, one might expect that these proteins perform similar functions for all herpesviruses. Previous studies on a UL16 null mutant derived from HSV-2 strain 186 revealed a roughly 100-fold replication defect and a critical role for UL16 in the nuclear egress of capsids. These findings were in stark contrast to what has been observed with UL16 mutants of HSV-1 and pseudorabies virus where roughly 10-fold replication deficiencies were reported that were accompanied by defects in the secondary envelopment of cytoplasmic capsids. One possible explanation for this discrepancy is that the HSV-2 186 strain is not representative of the HSV-2 species. To address this possibility, multiple UL16 null mutants were constructed in multiple HSV-2 and HSV-1 strains by CRISPR/Cas9 mutagenesis and their phenotypes characterized side-by-side. This analysis showed that all the HSV-2 UL16 mutants had 50 to 100-fold replication deficiencies that were accompanied by defects in the nuclear egress of capsids as well as defects in the secondary envelopment of cytoplasmic capsids. By contrast, most HSV-1 UL16 mutants had 10-fold replication deficiencies that were accompanied by defects in secondary envelopment of cytoplasmic capsids. These findings indicated that UL16 has HSV species-specific functions. Interestingly, HSV-1 UL16 could promote the nuclear egress of HSV-2 UL16 null strains, suggesting that, unlike HSV-1, HSV-2 lacks an activity that can compensate for nuclear egress in the absence of UL16.\n\nImportanceHSV-2 and HSV-1 are important human pathogens that cause distinct diseases in their hosts. A complete understanding of the morphogenesis of these viruses is expected to reveal vulnerabilities that can be exploited in the treatment of HSV disease. UL16 is a virion structural component that is conserved throughout the Herpesviridae and functions in virus morphogenesis, however, previous studies have suggested different roles for UL16 in the morphogenesis of HSV-2 and HSV-1. This study sought to resolve this apparent discrepancy by analyzing multiple UL16 mutant viruses derived from multiple strains of HSV-2 and HSV-1. The data indicate that UL16 has HSV species-specific functions insofar as HSV-2 has a requirement for UL16 in the escape of capsids from the nucleus whereas both HSV-2 and HSV-1 require UL16 for final envelopment of capsids at cytoplasmic membranes.

microbiology↗

bioRxiv2018

Differential splicing of ANP32A in birds alters its ability to stimulate RNA synthesis by restricted influenza polymerase

Adaptation of viruses to their host can result in specialization and a restricted host range. Species-specific polymorphisms in the influenza virus polymerase restrict its host range during transmission from birds to mammals. ANP32A was recently been identified as a cellular co-factor impacting polymerase adaption and activity. Avian influenza polymerases require ANP32A containing an insertion resulting from an exon duplication uniquely encoded in birds. Here we find that natural splice variants surrounding this exon create avian ANP32A proteins with distinct effects on polymerase activity. We demonstrate species-independent direct interactions between all ANP32A variants and the PB2 polymerase subunit. This interaction is enhanced in the presence of viral genomic RNA. In contrast, only avian ANP32A restored ribonucleoprotein complex assembly for a restricted polymerase by enhancing RNA synthesis. Our data suggest that ANP32A splicing variation amongst birds differentially impacts viral replication, polymerase adaption, and the potential of avian hosts to be reservoirs.

microbiology↗

bioRxiv2018

Analysis of Escherichia coli Flagellin Gene Expression in Simulated Spaceflight Growth Conditions

Population growth curves of Escherichia coli (E. coli) cultures propagated in spaceflight environments demonstrate an extended log phase duration and an increased final cell concentration compared to E. coli cultures grown on Earth. We suggest that during spaceflight, a lack of convective mixing in the growth medium alters a cells microenvironment such that the cell at first devotes its resources primarily to motility and later shifts most of its resources to reproduction. As flagellin is the major protein component of flagella, the motility structure of E. coli, we propose that the expression of the flagellin gene (fliC) can be used as a diagnostic marker for determining if the bacterium is in fact altering its allocation of cellular resources to motility during specific growth phases. We have used semiquantitative RT-PCR to analyze the expression of fliC in cultures grown in laboratory apparatus that simulate various aspects of the spaceflight environment. The data from this pilot study indicate that fliC expression increases initially under stationary, slow clinorotation, and fast clinorotation growth conditions, peaking at mid-exponential phase and decreasing dramatically in stationary phase. These data support the hypothesis that metabolic resources are being directed towards forming the flagellum, the bacteria motile structure, before shifting resources to growth, reproduction, and maintenance.

microbiology↗

bioRxiv2018

D,D-muramyl endopeptidase Spr contributes to intrinsic vancomycin resistance in Salmonella enterica serovar Typhimurium

The impermeability barrier provided by the outer membrane of enteric bacteria, a feature lacking in Gram-positive bacteria, plays a major role in maintaining resistance to numerous antimicrobial compounds and antibiotics. Here we demonstrate that mutational inactivation of spr, coding for a muramyl endopeptidase, significantly sensitizes Salmonella enterica serovar Typhimurium to vancomycin without any accompanying apparent growth defect or outer membrane destabilization. A similar phenotype was not achieved by deleting the mepA, pbpG, nlpC, yebA or yhdO genes coding for functional homologues to Spr. The spr mutant revealed increased autolytic behavior in response to not only vancomycin, but also to penicillin G, an antibiotic for which the mutant displayed a wild-type MIC. A screen for suppressor mutations of the spr mutant phenotype revealed that deletion of tsp (prc), encoding a periplasmic carboxypeptidase involved in processing Spr and PBP3, restored intrinsic resistance to vancomycin and reversed the autolytic phenotype of an spr mutant. Our data suggest that Spr contributes to intrinsic antibiotic resistance in S. Typhimurium without directly affecting the outer membrane permeability barrier. Furthermore, our data suggests that compounds that target specific cell wall endopeptidases might have the potential to expand the activity spectrum of traditional Gram-positive antibiotics.

microbiology↗

bioRxiv2018

Orthogonal dietary niche enables reversible engraftment of a gut bacterial commensal

Interest in manipulating the gut microbiota to treat disease has led to a need for understanding how organisms can establish themselves when introduced into a host with an intact microbial community. While probiotic or prebiotic approaches typically lead to a transient pulse in an organisms abundance, persistent establishment of an introduced species may require alternative strategies. Here, we introduce the concept of orthogonal niche engineering in the gut, where we include a resource typically absent from the diet, seaweed, to establish a customized niche for an introduced organism. We show that in the short term, co-introduction of this resource at 1% in the diet along with an organism with exclusive access to this resource, B. plebeius DSM 17135, enables it to colonize at a median abundance of 1%, frequently increasing in abundance to 10 or more percent. We construct a mathematical model of the system to infer that B. plebeius competitively acquires endogenous resources. We provide evidence that it competes with native commensals to achieve its observed abundance. We observe a diet-dependent loss in seaweed responsiveness of B. plebeius in the long term and show the potential for IgA-mediated control of putative invaders by the immune system. These results point to the potential for diet-based intervention as a means to introduce target organisms, but also indicate potential modes for failure of this strategy in the long term.

microbiology↗

bioRxiv2018

A dengue monovalent vaccine with novel structure provides cross-protection against four serotypes of dengue virus

Dengue fever is caused by four different serotypes of dengue virus (DENV) which is the leading cause of worldwide arboviral diseases in humans. The vaccine candidates under development require a tetravalent immunogen to induce a balanced immunity against all four serotypes of dengue virus. Herein we show that mice vaccinated with highly matured virus-like particles derived from DENV serotype 2 (mD2VLP) can generate higher and broader neutralization antibodies (NtAbs) against all 4 serotypes of DENV through clonal expansion supported by hybridoma and B-cell repertoire analysis. The cryo-electron microscopy reconstruction showed that mD2VLP particles possess a T=1 icosahedral symmetry with a groove located within the E-protein dimers near the 2-fold vertices that exposed highly overlapping, cryptic neutralizing epitopes. Most importantly, maternally transferred antibodies derived from mD2VLP-vaccinated female mice protected suckling mice from lethal challenge by all four serotypes of DENV. Our results support the fact that a universal dengue vaccine that protects against all four serotypes of dengue viruses can be achieved by using an immunogen such as mD2VLP.

microbiology↗

bioRxiv2018

The Rumen Metatranscriptome Landscape Reflects Dietary Adaptation and Methanogenesis in Lactating Dairy Cows

Methane eructed by ruminant animals is a main contributor to greenhouse gas emissions and is solely produced by members of the phylum Euryarchaeota within the domain Archaea. Methanogenesis depends on the availability of hydrogen, carbon dioxide, methanol and acetate produced, which are metabolic products of anaerobic microbial degradation of feed-derived fibers. Changing the feed composition of the ruminants has been proposed as a strategy to mitigate methanogenesis of the rumen microbiota.\n\nWe investigated the impact of corn silage enhanced diets on the rumen microbiota of rumen-fistulated dairy cows, with a special focus on carbohydrate breakdown and methanogenesis. Metatranscriptome analysis of rumen samples taken from animals fed corn silage enhanced diets revealed that genes involved in starch metabolism were significantly more expressed while archaeal genes involved in methanogenesis showed lower expression values. The nutritional intervention also influenced the cross-feeding between Archaea and Bacteria.\n\nThe results indicate that the ruminant diet is important in methanogenesis. The diet-induced changes resulted in a reduced methane emission. The metatranscriptomic analysis provided insights into key underlying mechanisms and opens the way for new rational methods to further reduce methane output of ruminant animals.

microbiology↗

bioRxiv2018

Bacterial swarming reduces Proteus mirabilis and Vibrio parahaemolyticus cell stiffness and increases β-lactam susceptibility

Swarmer cells of the gram-negative pathogenic bacteria Proteus mirabilis and Vibrio parahaemolyticus become long (>10-100 m) and multinucleate during their growth and motility on polymer surfaces. We demonstrate increasing cell length is accompanied by a large increase in flexibility. Using a microfluidic assay to measure single-cell mechanics, we identified large differences in swarmer cell stiffness of (bending rigidity of P. mirabilis, 9.6 x 10-22 N m2; V. parahaemolyticus, 9.7 x 10-23 N m2) compared to vegetative cells (1.4 x 10-20 N m2 and 3.2 x 10-22 N m2, respectively). The reduction in bending rigidity (~3-15 fold) was accompanied by a decrease in the average polysaccharide strand length of the peptidoglycan layer of the cell wall from 28-30 to 19-22 disaccharides. Atomic force microscopy revealed a reduction in P. mirabilis peptidoglycan thickness from 1.5 nm (vegetative) to 1.0 nm (swarmer) and electron cryotomography indicated changes in swarmer cell wall morphology. P. mirabilis and V. parahaemolyticus swarmer cells became increasingly sensitive to osmotic pressure and susceptible to cell wall-modifying antibiotics (compared to vegetative cells)--they were ~30% more likely to die after 3 h of treatment with minimum inhibitory concentrations of the {beta}-lactams cephalexin and penicillin G. The adaptive cost of swarming is offset by the increase in cell susceptibility to physical and chemical changes in their environment, thereby suggesting the development of new chemotherapies for bacteria that leverage swarming for colonization of hosts and survival. ImportanceProteus mirabilis and Vibrio parahaemolyticus are bacteria that infect humans. To adapt to environmental changes, these bacteria alter their cell morphology and move collectively to access new sources of nutrients in a process referred to as swarming. We found that a change in the composition and thickness of the peptidoglycan layer of the cell wall makes swarmer cells of P. mirabilis and V. parahaemolyticus more flexible (i.e., reduced cell stiffness) and increases their sensitivity to osmotic pressure and cell-wall targeting antibiotics (e.g., {beta}-lactams). These results highlight the importance of assessing the extracellular environment in determining antibiotic doses and the use of {beta}-lactams antibiotics for treating infections caused by swarmer cells of P. mirabilis and V. parahaemolyticus.

microbiology↗