MicrobiologyBytes

The structural complexities of bacteria are becoming increasingly apparent. Gram-negative bacteria can be divided into several subcellular compartments. There are two aqueous compartments called the cytoplasm and the periplasm. The cytoplasm is enclosed by a phospholipid bilayer called the inner membrane (IM), which is itself surrounded by an asymmetric bilayer called the outer membrane (OM). The periplasm lies in the space between the IM and OM, and is home to the peptidoglycan cell wall (CW). Present throughout these compartments are proteins with diverse and important biological functions. Some of these proteins are membrane-embedded and allow the transfer of molecules between compartments. Others are soluble enzymes involved in metabolic reactions. Much work has been devoted toward understanding how each of these compartments is formed and maintained. This review focuses on a particular aspect of OM biogenesis, namely the assembly of integral outer membrane proteins (OMPs).

Making a beta-barrel: assembly of outer membrane proteins in Gram-negative bacteria. Curr Opin Microbiol. 03 Jan 2012
The outer membrane (OM) of Gram-negative bacteria is an essential organelle that serves as a selective permeability barrier by keeping toxic compounds out of the cell while allowing vital nutrients in. How the OM and its constituent lipid and protein components are assembled remains an area of active research. In this review, we describe our current understanding of how outer membrane proteins (OMPs) are delivered to and then assembled in the OM of the model Gram-negative organism Escherichia coli.

Tweet

DNA fragments encoding enzymes, transcriptional regulators and virulence factors are fluxing through bacterial taxonomic walls by horizontal gene transfer. These elements often endow environmental and clinical strains of bacteria with new properties, including an enhanced virulence. Lateral genetic exchange, particularly of drug tolerance genes has been recognized for a long time; however, our understanding of this phenomenon is limited. Ontology and phylogeny of laterally transferred genetic elements are difficult to investigate, let alone the predictions of their insertion sites in hosts chromosomes.

An outbreak of the lethal Escherichia coli in Europe in 2011 highlighted the shortcoming of our knowledge on the basic principles of evolutionary trends of new pathogens. The outbreak first occurred in Germany in May 2011 where a rare enterohemorrhagic strain Escherichia coli O104:H4 caused haemolytic-uremic syndrome. The infection spread fast through many other European countries and sickened thousands of people. The level of lethality associated with the production of Shiga toxin by the strain and its resistance against many antibiotics was significant. A number of isolates from this outbreak have been sequenced and annotated. Based on the unique combination of genomic features these strains were suggested to represent a new pathotype Entero-Aggregative-Haemorrhagic E. coli (EAHEC).

Mainstreams of Horizontal Gene Exchange in Enterobacteria: Consideration of the Outbreak of Enterohemorrhagic E. coli O104:H4 in Germany in 2011. (2011) PLoS ONE 6(10): e25702. doi:10.1371/journal.pone.0025702
Escherichia coli O104:H4 caused a severe outbreak in Europe in 2011. The strain TY-2482 sequenced from this outbreak allowed the discovery of its closest relatives but failed to resolve ways in which it originated and evolved. On account of the previous statement, may we expect similar upcoming outbreaks to occur recurrently or spontaneously in the future? The inability to answer these questions shows limitations of the current comparative and evolutionary genomics methods. The study revealed oscillations of gene exchange in enterobacteria, which originated from marine γ-Proteobacteria. These mobile genetic elements have become recombination hotspots and effective ‘vehicles’ ensuring a wide distribution of successful combinations of fitness and virulence genes among enterobacteria. Two remarkable peculiarities of the strain TY-2482 and its relatives were observed: i) retaining the genetic primitiveness by these strains as they somehow avoided the main fluxes of horizontal gene transfer which effectively penetrated other enetrobacteria; ii) acquisition of antibiotic resistance genes in a plasmid genomic island of β-Proteobacteria origin which ontologically is unrelated to the predominant genomic islands of enterobacteria. Oscillations of horizontal gene exchange activity were reported which result from a counterbalance between the acquired resistance of bacteria towards existing mobile vectors and the generation of new vectors in the environmental microflora. We hypothesized that TY-2482 may originate from a genetically primitive lineage of E. coli that has evolved in confined geographical areas and brought by human migration or cattle trade onto an intersection of several independent streams of horizontal gene exchange. Development of a system for monitoring the new and most active gene exchange events was proposed. Tweet

Carbapenems were the last β-lactams retaining near-universal anti-Gram-negative activity, but carbapenemases are spreading, conferring resistance. New Delhi metallo-β-lactamase (NDM) enzymes are the latest carbapenemases to be recognized and since 2008 have been reported worldwide, mostly in bacteria from patients epidemiologically linked to the Indian subcontinent, where they occur widely in hospital and community infections, and also in contaminated urban water. The main type is NDM-1, but minor variants occur. NDM enzymes are present largely in Enterobacteriaceae, but also in non-fermenters and Vibrionaceae. Dissemination predominantly involves transfer of the bla(NDM-1) gene among promiscuous plasmids and clonal outbreaks. Bacteria with NDM-1 are typically resistant to nearly all antibiotics, and reliable detection and surveillance are crucial.

E. coli is one of the most prevalent human pathogens, and the window of opportunity to control it from becoming widely resistant is rapidly closing. No vaccine is likely to become available and one that affects commensal gut strains would probably be undesirable, even though these might act as vectors of potent resistance, including NDM-1. Therefore, everything must be done now to prevent infections due to bacteria with NDM-1, otherwise infections as common as pyelonephritis might soon become life-threatening owing to the lack of effective treatment.

The emerging NDM carbapenemases. Trends Microbiol. Nov 9 2011

Tweet

Noncovalent biological interactions are commonly subjected to mechanical force, particularly when they are involved in adhesion or cytoskeletal movements. While one might expect mechanical force to break these interactions, some of them form so-called catch bonds that lock on harder under force, like a nanoscale finger-trap. The adhesive protein FimH, which is located at the tip of E. coli fimbriae, allows bacteria to bind to urinary epithelial cells in a shear-dependent manner, binding at high but not at low flow. Isolated fimbrial tips, consisting of elongated protein complexes with FimH at the apex, reproduce this behavior in vitro. Models of the fimbrial tip structure show that FimH is shaped like a hook that is normally rigid but opens under force, causing structural changes that lead to firm anchoring of the bacteria on the surface. In contrast, the more distal adaptor proteins of the fimbrial tip create a flexible connection of FimH to the rigid fimbria, enhancing the ability of the adhesin to move into position and form bonds with mannose on the surface. The entire tip complex forms a hook-chain, ideal for rapid and stable anchoring in flow.

The Bacterial Fimbrial Tip Acts as a Mechanical Force Sensor. 2011 PLoS Biol 9(5): e1000617. doi:10.1371/journal.pbio.1000617
There is increasing evidence that the catch bond mechanism, where binding becomes stronger under tensile force, is a common property among non-covalent interactions between biological molecules that are exposed to mechanical force in vivo. Here, by using the multi-protein tip complex of the mannose-binding type 1 fimbriae of Escherichia coli, we show how the entire quaternary structure of the adhesive organella is adapted to facilitate binding under mechanically dynamic conditions induced by flow. The fimbrial tip mediates shear-dependent adhesion of bacteria to uroepithelial cells and demonstrates force-enhanced interaction with mannose in single molecule force spectroscopy experiments. The mannose-binding, lectin domain of the apex-positioned adhesive protein FimH is docked to the anchoring pilin domain in a distinct hooked manner. The hooked conformation is highly stable in molecular dynamics simulations under no force conditions but permits an easy separation of the domains upon application of an external tensile force, allowing the lectin domain to switch from a low- to a high-affinity state. The conformation between the FimH pilin domain and the following FimG subunit of the tip is open and stable even when tensile force is applied, providing an extended lever arm for the hook unhinging under shear. Finally, the conformation between FimG and FimF subunits is highly flexible even in the absence of tensile force, conferring to the FimH adhesin an exploratory function and high binding rates. The fimbrial tip of type 1 Escherichia coli is optimized to have a dual functionality: flexible exploration and force sensing. Comparison to other structures suggests that this property is common in unrelated bacterial and eukaryotic adhesive complexes that must function in dynamic conditions.

Bacterial virulence results from the interaction between bacteria and their hosts. This interaction provides selection pressure for bacterial adaptation towards increased fitness or virulence. Basic mechanisms involved in bacterial adaptation at the genetic level are point mutations and recombination. As bacterial genome plasticity is higher in vivo than in vitro, host-pathogen interaction may facilitate bacterial adaptation. Comparative genomics has so far been almost entirely focused on genomic changes upon prolonged bacterial growth in vitro.

To achieve a better comprehension of bacterial genome plasticity and the capacity to adapt in response to their host, researchers studied bacterial genome evolution in vivo. They analyzed the impact of individual hosts on genome-wide bacterial adaptation under controlled conditions, by administration of an asymptomatic E. coli isolate to several hosts. Interestingly, the different hosts appeared to personalize their microflora. Adaptation at the genomic level included point mutations in several metabolic and virulence-related genes, often affecting pleiotropic regulators, but re-isolates from each patient showed a distinct pattern of genetic alterations in addition to random changes. These results provide new insights into bacterial traits under selection during E. coli in vivo growth, further explaining the mechanisms of bacterial adaptation to specific host environments.

Host Imprints on Bacterial Genomes – Rapid, Divergent Evolution in Individual Patients. (2010) PLoS Pathog 6(8): e1001078. doi:10.1371/journal.ppat.1001078
Bacterial virulence results from the interaction between bacteria and their hosts. This interaction provides selection pressure for bacterial adaptation towards increased fitness or virulence. Basic mechanisms involved in bacterial adaptation at the genetic level are point mutations and recombination. As bacterial genome plasticity is higher in vivo than in vitro, host-pathogen interaction may facilitate bacterial adaptation. Comparative genomics has so far been almost entirely focused on genomic changes upon prolonged bacterial growth in vitro. To achieve a better comprehension of bacterial genome plasticity and the capacity to adapt in response to their host, we studied bacterial genome evolution in vivo. We analyzed the impact of individual hosts on genome-wide bacterial adaptation under controlled conditions, by administration of asymptomatic bacteriuria E. coli isolate 83972 to several hosts. Interestingly, the different hosts appeared to personalize their microflora. Adaptation at the genomic level included point mutations in several metabolic and virulence-related genes, often affecting pleiotropic regulators, but re-isolates from each patient showed a distinct pattern of genetic alterations in addition to random changes. Our results provide new insights into bacterial traits under selection during E. coli in vivo growth, further explaining the mechanisms of bacterial adaptation to specific host environments.

Related:

• The long now – 40,000 generations of E. coli
• Spread of Pathogenicity Islands in Escherichia coli
• The continuing evolution of E. coli O157:H7

The dense bacterial consortium called the “microbiota” that inhabits the intestinal tract is recognized increasingly as playing a major role in human health and disease. The microbiota generally influences the host in a beneficial fashion by shaping gastrointestinal and immune functions, exerting protection against pathogens, and contributing to metabolic pathways. Escherichia coli is a consistent member of the humanintestinal microbiota, colonizing the intestine within a few days after birth and persisting throughout the life of the host. The E. coli strain population can be categorized in at least four major phylogenetic groups, each group being more specifically associated with certain ecological niches. E. coli strains belonging to group B2 are recovered from the environment less frequently but can persist longer in the colon than other groups and represent 30–50% of strains isolated from the feces of healthy humans living in high-income countries.

Up to 34% of commensal E. coli strains of the phylogenetic group B2 carry a conserved genomic island named the “pks island”. This gene cluster codes for genes that allow production of a putative hybrid peptide-polyketide genotoxin, Colibactin. In vitro infection with these strains induces DNA double-strand breaks (DSBs) in cultivated human cells, but the pks island was not proved to cause DNA damage in vivo. In this study, the authors explore whether those bacteria were able to induce genetic damage in vivo on the colonic mucosa and to characterize the consequences of this damage on mammalian cells in relation with the number of infecting bacteria. They report that pks+ E. coli induced DSBs in vivo. In addition, infection of various mammalian cells with pks+ E. coli induced, at very low multiplicity of infection, reversible DNA damage response that did not repair all DSBs, leading to chronic mitotic and chromosomal aberrations together with increased frequency of gene mutation and anchorage-independent growth. Taken together, these findings strongly suggest that these pks+ strains are genotoxic in vivo and provide insights into mechanisms by which common E. coli strains may contribute to cellular transformation and possibly sporadic colorectal cancer tumorigenesis.

Escherichia coli induces DNA damage in vivo and triggers genomic instability in mammalian cells. PNAS USA June 7 2010 doi: 10.1073/pnas.100126110
Escherichia coli is a normal inhabitant of the human gut. However, E. coli strains of phylogenetic group B2 harbor a genomic island called “pks” that codes for the production of a polyketide-peptide genotoxin, Colibactin. Here we report that in vivo infection with E. coli harboring the pks island, but not with a pks isogenic mutant, induced the formation of phosphorylated H2AX foci in mouse enterocytes. We show that a single, short exposure of cultured mammalian epithelial cells to live pks+ E. coli at low infectious doses induced a transient DNA damage response followed by cell division with signs of incomplete DNA repair, leading to anaphase bridges and chromosome aberrations. Micronuclei, aneuploidy, ring chromosomes, and anaphase bridges persisted in dividing cells up to 21 d after infection, indicating occurrence of breakage–fusion–bridge cycles and chromosomal instability. Exposed cells exhibited a significant increase in gene mutation frequency and anchorage-independent colony formation, demonstrating the infection mutagenic and transforming potential. Therefore, colon colonization with these E. coli strains harboring the pks island could contribute to the development of sporadic colorectal cancer.

Related:

• Spread of Pathogenicity Islands in Escherichia coli
• The continuing evolution of E. coli O157:H7
• Global functional atlas of Escherichia coli proteins

Swarming is flagella-driven bacterial group motility over a surface, which is observed in the laboratory on media solidified with agar. The percentage of agar is critical for enabling swarming. Some bacteria like Vibrio parahaemolyticus and Proteus mirabilis can swarm readily on higher percentage agar, whereas others like Salmonella, Escherichia coli, Serratia, Pseudomonas, and Bacillus swarm only on lower percentage agar. Hard-agar swarmers differentiate into specialized swarm cells that are elongated and have increased flagella. Medium-agar swarmers generally do not display a similar differentiated morphology. In many of the latter class of swarmers (e.g. Serratia, Pseudomonas, Bacillus), movement is enabled by powerful extracellular surfactants whose synthesis is under quorum-sensing control. Surfactants lower surface tension and allow rapid colony expansion. Salmonella and E. coli do not appear to make such surfactants.

Swarming bacteria exhibit adaptive resistance to multiple antibiotics. Analysis of this phenomenon has revealed the protective power of high cell densities to withstand exposure to otherwise lethal antibiotic concentrations. This paper shows that that high cell densities promote bacterial survival, even in a nonswarming state, but that the ability to move, as well as the speed of movement, confers an added advantage, making swarming an effective strategy for prevailing against antimicrobials. There is no evidence of induced resistance pathways or quorum-sensing mechanisms controlling this group resistance, which occurs at a cost to cells directly exposed to the antibiotic. This work is relevant to the adaptive antibiotic resistance of bacterial biofilms.

Cell density and mobility protect swarming bacteria against antibiotics. PNAS USA February 2 2010 doi: 10.1073/pnas.0910934107

Related:

• Look who’s talking
• Antibiotics, your gut, and you
• MicrobiologyBytes: Antibiotics

A 21-year Michigan State University experiment that distills the essence of evolution in laboratory flasks not only demonstrates natural selection at work, but could lead to biotechnology and medical research advances. Researchers at Michigan State University started a culture of Escherichia coli bacteria in 1988. The results are in.

Genome evolution and adaptation in a long-term experiment with Escherichia coli. Nature 18 October 2009 doi:10.1038/nature08480
The relationship between rates of genomic evolution and organismal adaptation remains uncertain, despite considerable interest. The feasibility of obtaining genome sequences from experimentally evolving populations offers the opportunity to investigate this relationship with new precision. Here we sequence genomes sampled through 40,000 generations from a laboratory population of Escherichia coli. Although adaptation decelerated sharply, genomic evolution was nearly constant for 20,000 generations. Such clock-like regularity is usually viewed as the signature of neutral evolution, but several lines of evidence indicate that almost all of these mutations were beneficial. This same population later evolved an elevated mutation rate and accumulated hundreds of additional mutations dominated by a neutral signature. Thus, the coupling between genomic and adaptive evolution is complex and can be counterintuitive even in a constant environment. In particular, beneficial substitutions were surprisingly uniform over time, whereas neutral substitutions were highly variable.

Related:

• The continuing evolution of E. coli O157:H7
• Global functional atlas of Escherichia coli proteins

The urinary tract is among the most common sites of bacterial infection. Over half (53%) of all women and 14% of men experience at least one urinary tract infection (UTI) in their lifetime, leading to an average of 6.8 million physician office visits, 1.3 million emergency room visits, and 245,000 hospitalizations per year, with an annual cost of over US$2.4 billion in the United States alone. Escherichia coli is the infectious agent in more than 80% of uncomplicated UTIs, which occur in patients with a normal urinary tract devoid of structural abnormalities or inflammatory lesions.

In light of the recent E. coli outbreak in the UK, the news that scientists have made an important step toward what could become the first vaccine to prevent urinary tract infections is interesting. To help combat this common health issue, the scientists used a novel systematic approach combining bioinformatics, genomics and proteomics to look for key parts of the E. coli bacterium that could be used in a vaccine to elicit an effective immune response. The team screened 5,379 possible bacterial proteins and identified three strong candidates to use in a vaccine to prime the body to fight E. coli, the cause of most uncomplicated urinary tract infections. The vaccine produced prevented infection and produced key types of immunity when tested in mice.

Scientists have attempted to develop a vaccine for UTIs over the past two decades. This latest potential vaccine has features that may better its chances of success. It alerts the immune system to iron receptors on the surface of bacteria that perform a critical function allowing infection to spread. Administered via the nose rather than injected, it induces an immune response in the body’s mucosa, a first line of defense against invading pathogens. The protective immune response, which also produced in mucosal tissue in the urinary tract, should help the body fight infection where it starts. The research team is currently testing more strains of E. coli. Most of the strains produce the same iron-related proteins that the vaccine targets, an encouraging sign that the vaccine could work against many urinary tract infections.

Iron acquisition is a critical function required by bacteria in order to cause infections. In uropathogenic E. coli, this function is mediated by a repertoire of systems that scavenge iron from the host during infection. Vaccination with certain iron receptors from these systems is sufficient to elicit protective immunity from experimental urinary tract infection. Induction of an antibody response played a key role in protection from infection because antibody class-switching and the production of antibodies in urine correlated with reduced numbers of bacteria in the bladder. By targeting an entire class of molecules involved in iron acquisition instead of a single protein, it was possible to successfully identify components of a protective UTI vaccine. This strategy could be a useful approach in the development of vaccines to prevent infections caused by other pathogenic bacteria.

However, this is still early clinical research and this candidate vaccine has not yet undegone even a phase 1 safety trial in humans. And even if that were successful, the vaccine would take several more years to reach the market, even if manufacturers decided they could make a profit from producing it. So the next time you’re down on the farm, wash your hands – and make sure that burger is properly cooked through.

Mucosal Immunization with Iron Receptor Antigens Protects against Urinary Tract Infection. 2009PLoS Pathog 5(9): e1000586 doi:10.1371/journal.ppat.1000586
Uncomplicated infections of the urinary tract, caused by uropathogenic Escherichia coli, are among the most common diseases requiring medical intervention. A preventive vaccine to reduce the morbidity and fiscal burden these infections have upon the healthcare system would be beneficial. Here, we describe the results of a large-scale selection process that incorporates bioinformatic, genomic, transcriptomic, and proteomic screens to identify six vaccine candidates from the 5379 predicted proteins encoded by uropathogenic E. coli strain CFT073. The vaccine candidates, ChuA, Hma, Iha, IreA, IroN, and IutA, all belong to a functional class of molecules that is involved in iron acquisition, a process critical for pathogenesis in all microbes. Intranasal immunization of CBA/J mice with these outer membrane iron receptors elicited a systemic and mucosal immune response that included the production of antigen-specific IgM, IgG, and IgA antibodies. The cellular response to vaccination was characterized by the induction and secretion of IFN-c and IL-17. Of the six potential vaccine candidates, IreA, Hma, and IutA provided significant protection from experimental infection. In immunized animals, class-switching from IgM to IgG and production of antigen-specific IgA in the urine represent immunological correlates of protection from E. coli bladder colonization. These findings are an important first step toward the development of a subunit vaccine to prevent urinary tract infections and demonstrate how targeting an entire class of molecules that are collectively required for pathogenesis may represent a fundamental strategy to combat infections.

Related:

• Microbiology Video Library: Escherichia coli
• E. coli O157:H7 – getting to the bottom of the burger bug
• The continuing evolution of E. coli O157:H7
• Bacterial toxins