Category Archives: Microbiology

All hail the new hepatitis C mouse model

With existing treatments only partially effective with major adverse effects and no vaccine currently available, hepatitis C virus (HCV) infection is a major health problem worldwide. An estimated 120 million people are chronically infected around the world and, therefore, at increased risk of liver damage (fibrosis and cirrhosis) and liver cancer. Research into potential new vaccines and therapies for HCV has been severely hampered by the lack of a small animal mouse model, often a crucial research tool to investigate disease progression and to test new drugs. Now, US scientists have for the first time made a genetically humanized mouse model for hepatitis C, which could prove vital in HCV infection research.
HCV is spread via blood-to-blood contact; anything from blood transfusions, sharing contaminated needles in injection drug use, and, as Pamela Anderson found out, contaminated tattoo needles. Diagnosis can be problematic and disease progression can be unpredictable, infected individuals range from being asymptomatic, to clearing the virus naturally or suffering progressive liver damage that, ultimately, leads to liver failure and need for transplantation.
Mice are normally resistant to HCV infection, only humans and chimpanzees are naturally permissive to HCV, and at least four human factors are critical for HCV entry, claudin 1, occuldin, CD81 and scavenger receptor type B class I (SCARBI). In their paper published this week in Nature, Marcus Dorner and colleagues built on existing knowledge that in vitro rodent cells only need to express occludin and CD81 to enable HCV entry. They reasoned that expressing these key human genes (CD81 and occludin) in mice could make living animals susceptible to HCV infection.
The scientists made mice that expressed human SCARB1, claudin 1, occludin and CD81 using an adenovirus as a vector to deliver the human genes into the mouse liver. Although mouse liver cells expressed these human genes (5% of cells expressed all four genes, whilst 18–25% expressed both CD81 and occludin), infecting these mice with HCV and proving they were infected was the major stumbling block as HCV infection in murine cells in vitro and in vivo is inefficient. Even though mice were infected with bioluminescent HCV (tagged with firefly luciferase), which can be easily detected if they replicated (the cells would ‘glow’), bioluminescent signals were not above background levels making it difficult to detect the virus. As an alternative approach, the mice were engineered to express the luciferase reporter whilst the HCV genome was engineered to express a protein that activates the bioluminesce reporter gene, such that delivery and replication of HCV in the liver leads to a bioluminescent signal. In this way, the researchers showed that all mice expressing at least human occludin and CD81 could indeed be infected with HCV. They then went on to validate their new model and demonstrated the in vivo role of SCARB1 in viral entry and uptake into host cells. Furthermore, the study authors managed to block HCV entry using passive immunisation (transfer of readymade anti-HCV antibodies) in the humanized mice. A promising HCV vaccine candidate (a recombinant vaccine virus vector expressing HCV proteins that has been shown to work in chimps) was also tested in the model mice and was shown to induce immunity and partial protection against HCV infection.
“To our knowledge, this is the first time that any step in the viral life cycle has been recapitulated in a rodent simply by the expression of human genes,” write the study authors. This new mouse model should enable scientists to closely study hepatitis C disease progression in a small animal model that is more amenable to lab research. Hopefully, new improved strategies (both drugs and vaccines) against HCV can be developed and used to guide any future clinical trials.

 

ResearchBlogging.orgDorner, M., Horwitz, J., Robbins, J., Barry, W., Feng, Q., Mu, K., Jones, C., Schoggins, J., Catanese, M., Burton, D., Law, M., Rice, C., & Ploss, A. (2011). A genetically humanized mouse model for hepatitis C virus infection Nature, 474 (7350), 208-211 DOI: 10.1038/nature10168

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Microbial forensics: the science behind the Amerithrax investigation

Nearly a decade after the postal anthrax attacks in the USA that killed 5 individuals and infected more than 20 people, scientists have revealed the measures used to trace the Bacillus anthracis strain used in the bioterror attack in a new paper available online for free from Proceedings of the National Academy of Sciences. A groundbreaking mix of genomics and microbiology were used as part of the criminal investigation  into the 2001 anthrax attacks (called Amerithrax); microbial forensics proved key to identifying the exact flask from which the anthrax spores were taken.

Rasko and colleagues used highly accurate whole-genome sequencing and comparative genomics (against the B. anthracis Ames ancestor, believed to be the progenitor of all Ames lab samples and used as a gold standard reference strain in the USA) to determine the source strain of B. anthracis used in the letter attacks. First, the scientists took spore samples from some of the letters and grew them in the lab. A number of morphological variants were observed in these letter-isolated bacterial samples (yellow or yellow–grey coloured rather than the usual grey–white of wild-type anthrax colonies) and all had diminished abilities to sporulate. These variants were then sequenced and compared with genomes sequences of the gold standard Ames ancestor to identify four distinct loci with genetic mutations (three of which were in B. anthracis sporulation pathways, specifically regulation of a key protein, Spo0F) in the morpholigical variants—features unique to the isolated anthrax variants. None of these variants were found to be prevalent in the environment (even in the areas associated with the Amerithrax investigation).

Ultimately, using comparisons with genomes of repository anthrax sources, the anthrax spores used to lace the letters were found to have a unique genetic fingerprint; anthrax batches were eventually traced back to a source flask (RMR-1029) in the lab of Dr Bruce Ivins (a key suspect in the subsequent criminal investigation who later committed suicide before a criminal case could be brought to trial).

The study authors conclude that the B. anthracis bioterror attack investigations “taught us important lessons about the integration of whole-genome sequencing for forensic applications”, although they do concede that their methods might not applicable to other bioterror agents.

ResearchBlogging.orgRasko, D., Worsham, P., Abshire, T., Stanley, S., Bannan, J., Wilson, M., Langham, R., Decker, R., Jiang, L., Read, T., Phillippy, A., Salzberg, S., Pop, M., Van Ert, M., Kenefic, L., Keim, P., Fraser-Liggett, C., & Ravel, J. (2011). Bacillus anthracis comparative genome analysis in support of the Amerithrax investigation Proceedings of the National Academy of Sciences DOI: 10.1073/pnas.1016657108

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Diagnosing schistosomiasis: urine-based tests better than traditional assays?

Taken from Wikipedia

Intestinal schistosomiasis (caused by the parasite Schistosoma mansoni) can be detected easily and accurately by measuring levels of an excreted parasite antigen called circulating cathodic antigen, or CCA, in an individual’s urine, according to new research published in PLoS Neglected Tropical Diseases. This method has been found to be at least as effective as the existing ‘gold standard’ diagnostic test the Kato-Katz assay—literally counting the number of parasite eggs per gram of poo—and also to blood tests that test for parasite-specific antibodies. Moreover, switching to these urine-based tests for schistomiasis diagnosis is, quite frankly, preferable to sifting through someone’s faeces (messy, risky, time consuming, and actually not that useful), and could also make it easier to assess the prevalence and resolution of S. mansoni infection, which could be beneficial for disease control programmes.

Taking advantage of a large study to assess schistosomiasis prevalence in young children, the researchers tested the sensitivity and specificity of two different types of CCA urine tests—a strip test designed for use in the laboratory (unfortunately no longer under production) and a cassette test suited for field use—and compared them to both the Kato-Katz technique for assessing S. mansoni egg burden and ELISAs to detect anti-schistosome antibodies. Stool, urine and blood samples were collected from 484 children (aged 1–15 years old) who lived in the village, Usoma, in Western Kenya, which is near Lake Victoria, an area known to have high S. mansoni infection rates. Even when taking into account the limitations of the Kato-Katz assay in their analysis, the urine-based diagnostics tests were still sensitive and specific with CCA test levels reflecting the stool egg burden, and thus the intensity of the infection (a finding that confirmed other study results). Moreover, the tests worked even if the children were also infected with other parasites (Ascaris lumbricoides, hookworm or Trichuris trichuria), which can affect the performance of some diagnostic tests.

The CCA diagnostic test for schistomiasis has now been shown to be effective in areas with a high burden of the disease, but more work is needed to see whether it is sensitive enough to detect disease in areas with low S. mansoni infection rates. Whether these urine tests will eventually replace the tried and tested Kato-Katz technique remains to be seen; one advantage of examining stool samples is that other parasites may be found and identified at the same time, which is not possible with the CCA test as it is specific for schistosomiasis.

ResearchBlogging.orgShane, H., Verani, J., Abudho, B., Montgomery, S., Blackstock, A., Mwinzi, P., Butler, S., Karanja, D., & Secor, W. (2011). Evaluation of Urine CCA Assays for Detection of Schistosoma mansoni Infection in Western Kenya PLoS Neglected Tropical Diseases, 5 (1) DOI: 10.1371/journal.pntd.0000951

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CFTR aids Listeria escape into cell cytosol

Courtesy of CDC/ Dr. Balasubr Swaminathan; Peggy Hayes

The intracellular pathogen Listeria monocytogenes must escape the vacuole formed during entry into the host cell to replicate in its preferred environment—the cell cytosol—and continues its life cycle. Although the pore-forming bacterial toxin listeriolysin O is vital for Listeria escape and virulence, new research by Radtke and colleagues published online in PNAS shows that a host cell protein, CFTR (cystic fibrosis transmembrane conductance regulator, which forms a chloride ion channel that, incidentally, when dysfunctional results in cystic fibrosis), promotes escape of L. monocyotgenes from intracellular vacuoles.

Radtke et al. reasoned that, as the intravacuolar environment is dynamic and likely modulated by a variety of proteins, regulation of ion flux whilst Listeria is inside a vacuole could affect its subsequent escape from this membrane-bound organelle. The researchers confirmed that CFTR was endogenously expressed by mouse macrophages and addition of a CFTR inhibitor did not affect uptake of Listeria into host cells but did reduce the number of intracellular bacteria, indicating that the bacteria might be trapped within the vacuole. Using macrophages isolated from either wild-type mice or mice carrying the CFTR mutation associated with human cystic fibrosis, they found that defects in CFTR led to delayed intracellular replication (indicative of a defect in vacuole escape). Finally, the researchers conclude that CFTR potentially promotes escape of Listeria by controlling the flux of chlorides into the vacuole—a high chloride concentration seems to increase both the oligomerisation and haemolytic activity of listeriolysin O, the key bacterial toxin needed for escape.

 

Little is known about the role of ion transport in the context of bacterial infection and it would be interesting to see whether other ion channels and transporters also contribute to the virulence of Listeria and other intracellular bacteria.

 

 ResearchBlogging.orgRadtke, A., Anderson, K., Davis, M., DiMagno, M., Swanson, J., & O’Riordan, M. (2011). Listeria monocytogenes exploits cystic fibrosis transmembrane conductance regulator (CFTR) to escape the phagosome Proceedings of the National Academy of Sciences DOI: 10.1073/pnas.1013262108

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Spreading Salmonella—hyper-replicating bacteria act as a reservoir for dissemination

New research reveals how Salmonella enterica spread in the gut and gallbladder—a subpopulation of Salmonella primed for invasion rapidly replicate in the host cell cytosol such that bacteria-laden cells are extruded out of the epithelial-cell layer releasing invasive Salmonella into the gastrointestinal and biliary lumen. Leigh Knodler and colleagues write that other mucosal-dwelling pathogens could use this “host cell process as an exit strategy”.

Salmonella species can cause a range of infections from typhoid fever to food poisoning. Ordinarily, the intracellular bacteria Salmonella enterica resides and replicates within a membrane-bound vacuole in epithelial cells. During its life cycle, the bacteria are adapted to survive within a wide range of environmental niches within the human host (including cells such as enterocytes and macrophages and organs such as the spleen and gastrointestinal tract).

Knodler et al. observed a subpopulation of Salmonella that were ‘hyper-replicating’; these bacteria were doubling in number at almost five times the rate of the overall population of bacteria in the epithelial cell. Not only that, these bacteria were rapidly proliferating not in the Salmonella-containing vacuole, but in the host cell cytosol (which is believed to be nutrient rich) and were ready to invade other cells (they expressed type III secretion system 1 components and flagella, virulence factors that are required for invasion). Moreover, epithelial cells overloaded with these hyper-replicating cytosolic Salmonella were forced out of the apical side of the epithelial-cell layer—just as when dying cells are extruded out of the epithelium during the normal rapid turnover of epithelial cells that occurs to maintain the gut epithelium. Subsequently, invasive bacteria are released into the lumen and are primed and ready to infect new cells. The extruded host cells then die in a caspase-1-dependent manner and trigger the production of the proinflammatory cytokine interleukin 18—a process which could, in part, explain the high levels of mucosal inflammation observed in Salmonella infections of the gut and gallbladder.


ResearchBlogging.orgKnodler, L., Vallance, B., Celli, J., Winfree, S., Hansen, B., Montero, M., & Steele-Mortimer, O. (2010). Dissemination of invasive Salmonella via bacterial-induced extrusion of mucosal epithelia Proceedings of the National Academy of Sciences, 107 (41), 17733-17738 DOI: 10.1073/pnas.1006098107

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Walking with bacteria

They swim, they swarm, they twitch and glide…they even ride on comet tails, and now it seems that bacteria can ‘walk’ as Maxsim Gibiansky and colleagues demonstrate in their short but sweet research published in Science.

Gibiansky et al. studied the behaviour of Pseudomonas aeruginosa, a bacteria that is ordinarily found in soil and water, but has increasingly been associated with opportunistic infections in humans (and is a particular problem in those with cystic fibrosis). A key feature of P. aeruginosa is that these bacteria form multicellular, surface-bound communities called biofilms and are able to move within these communities by twitching motility owing to their type IV pili (hair-like structures on bacteria that can extend, tether to a surface and then retract to move bacteria along). The researchers studied microscopy movies of the P. aeruginosa biofilms and used computer software to track how the bacteria transitioned from planktonic state (that is, freely suspended in liquid) to the surface-bound biofilm.

Two different surface motility mechanisms were observed just after P. aeruginosa bacteria attached to a surface, but before a microcolony of bacteria were formed. The scientists studied mutant bacteria lacking flagella (a tail-like bacterial appendage that can also enable bacteria to move) that can only move using their type IV pili. These bacteria tended to ‘crawl’ in one direction when positioned horizontal to the surface and ‘walked’ in all directions when attached vertically to the surface by one end of the bacteria. Each movement mechanism was useful for surface exploration; crawling enabled directional movement across larger areas (6 μm distance) than walking, which enabled rapid exploration in local areas (up to 2 μm distance). Furthermore, these same movements were observed in wild-type bacteria. Moreover, the orientation of bacteria influenced biofilm morphology. Surface detachment was facilitated by type IV pili by tilting bacteria from horizontal to vertical positions and after bacterial division newborn bacteria detach and then ‘walk’ away. Finally, bacteria lacking type IV pili could neither ‘crawl’ or ‘walk’.

Scientific observations like this brevia report add to the understanding of bacterial behaviour in biofilms and could eventually lead to useful, new treatments against biofilm-forming pathogens.

 

ResearchBlogging.orgGibiansky, M., Conrad, J., Jin, F., Gordon, V., Motto, D., Mathewson, M., Stopka, W., Zelasko, D., Shrout, J., & Wong, G. (2010). Bacteria Use Type IV Pili to Walk Upright and Detach from Surfaces Science, 330 (6001), 197-197 DOI: 10.1126/science.1194238

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Altruistic bacterial charity workers help protect their vulnerable stressed out kin

US scientists have found that a small minority of highly antibiotic-resistant bacteria will produce and share a molecule, indole, that can activate survival mechanisms in less-resistant cells to enable the whole bacterial population to survive stressful environments despite the fact that production of this signalling molecule weakens the fitness of bacteria.

The increasing incidence of antibiotic resistance and the emergence of so-called ‘superbugs’ are of huge importance to medicine and society as a whole with the ever-increasing likelihood of a return to a world without antibiotics. This potentially disastrous public health crisis led the Infectious Diseases Society of America to launch the “10x’20” initiative in which they call for a global commitment to research and develop 10 new, effective antibiotic drugs by 2020. As a complement to this drug development, research into how bacteria develop this resistance could provide crucial clues for the rational design of new antimicrobial agents.

Henry Lee and colleagues investigated the population dynamics of antibiotic resistance. They grew a vat of E. coli with increasing amounts of the antibiotic norfloxacin and then took samples of the bacteria and monitored the percentage of bacteria that became resistant to the antibiotic. The scientists found an individual isolate that was highly resistant to norfloxacin (even higher than the greatest norfloxacin levels tested in their bioreactor). These bacteria produced indole, which is known to aid tolerance to stress in E. coliindole induces anti-stress mechanisms such as drug efflux pumps that help drive out toxic substances from the bacterial cell—although its production can reduce the overall fitness of the bacteria. Moreover, indole boosts the antibiotic resistance of the whole bacterial population and not just the select few that produce it. This population-based resistance was not drug specific and was even observed when the scientists challenged E. coli with gentamicin, which is a different type of antibiotic (with a different mode of action) to the quinolone norfloxacin.

The researchers conclude that under antibiotic stress, a few drug-resistant mutants will endure a fitness cost to produce and share the benefits of the metabolite indole to “shield the less-resistant bacteria from antibiotic insult” and enable these ‘weak’ bacteria to survive.

ResearchBlogging.orgLee HH, Molla MN, Cantor CR, & Collins JJ (2010). Bacterial charity work leads to population-wide resistance. Nature, 467 (7311), 82-5 PMID: 20811456

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