MicrobiologyBytes

Mycobacterium tuberculosis was first isolated more than 125 years ago. Although a huge amount of research has been devoted to it over this time, tuberculosis still represents a major public health threat in many countries. The main hindrances in fighting this disease include a lack of understanding of the human infection, its establishment and progression, as well as the host-pathogen interactions that determine the different outcomes. Furthermore, the treatment regimen of six months administration of up to four drugs has not evolved in more than four decades, and recent years have seen an alarming increase in multi-drug resistant (MDR) and extensively drug-resistant (XDR) strains. It is clear then that novel and imaginative approaches are needed to speed up both basic and translational research in tuberculosis, especially in the areas of vaccine and drug development.

Optimisation of Bioluminescent Reporters for Use with Mycobacteria. 2010 PLoS ONE 5(5): e10777. doi:10.1371/journal.pone.0010777
Mycobacterium tuberculosis, the causative agent of tuberculosis, still represents a major public health threat in many countries. Bioluminescence, the production of light by luciferase-catalyzed reactions, is a versatile reporter technology with multiple applications both in vitro and in vivo. In vivo bioluminescence imaging (BLI) represents one of its most outstanding uses by allowing the non-invasive localization of luciferase-expressing cells within a live animal. Despite the extensive use of luminescent reporters in mycobacteria, the resultant luminescent strains have not been fully applied to BLI.
One of the main obstacles to the use of bioluminescence for in vivo imaging is the achievement of reporter protein expression levels high enough to obtain a signal that can be detected externally. Therefore, as a first step in the application of this technology to the study of mycobacterial infection in vivo, we have optimised the use of firefly, Gaussia and bacterial luciferases in mycobacteria using a combination of vectors, promoters, and codon-optimised genes. We report for the first time the functional expression of the whole bacterial lux operon in Mycobacterium tuberculosis and M. smegmatis thus allowing the development of auto-luminescent mycobacteria. We demonstrate that the Gaussia luciferase is secreted from bacterial cells and that this secretion does not require a signal sequence. Finally we prove that the signal produced by recombinant mycobacteria expressing either the firefly or bacterial luciferases can be non-invasively detected in the lungs of infected mice by bioluminescence imaging.
While much work remains to be done, the finding that both firefly and bacterial luciferases can be detected non-invasively in live mice is an important first step to using these reporters to study the pathogenesis of M. tuberculosis and other mycobacterial species in vivo. Furthermore, the development of auto-luminescent mycobacteria has enormous ramifications for high throughput mycobacterial drug screening assays which are currently carried out either in a destructive manner using LuxAB or the firefly luciferase.

Related:

• Public health and bovine tuberculosis
• Origin and spread of Mycobacterium tuberculosis
• Tuberculosis – is the white plague winning?

The Astroviridae is a family of nonenveloped, positive-sense single-stranded RNA viruses. They are classified into mamastroviruses and avastroviruses and are known to infect mammalian and avian species, respectively. In humans, the classical human astroviruses are genetically closely related and can be classified into 8 serotypes (HAstV1-8). In addition, several genetically distinct human astroviruses (e.g. MLB1) have been recently identified in stool samples from patients suffering from gastroenteritis. Although astroviruses are one of the major causative agents of gastroenteritis, there is relatively little information on the ecology and evolution of these viruses. The recent discoveries of genetically diverse astroviruses in bats and other animals highlights the genetic diversity of astroviruses in nature and suggest that there might be many more novel astroviruses circulating in peri-domestic and wild animals. Bats represent the second largest group of mammals (comprises 20% of all mammals). The remarkably high detection rate of astroviruses in bats prompted researchers to perform a surveillance study on rodents, which are the largest group of mammals accounting for 40% of known mammalian species. They specifically targeted rodent species that have a long history of living in close proximity to human populations. This paper describes the discovery of rat astroviruses (RAstVs) from urban rats.

Detection of novel astroviruses in urban brown rats and previously known astroviruses in humans. J Gen Virol. Jun 16 2010
Several novel astroviruses have been recently discovered in humans and in other animals. Here, we report results from our surveillance of astroviruses in human and rodent fecal samples in Hong Kong. Classical human astroviruses (N=9) and a human MLB1 astrovirus were detected in human fecal samples (N=622). Novel astroviruses were detected from 1.6% of the fecal samples of urban brown rat (Rattus norvegicus) (N=441), indicating the prevalence of astrovirus infection in rats might be much lower to those recently observed in bats. These rat astroviruses were phylogenetically related to recently discovered human astroviruses MLB1 and MLB2, suggesting the MLB viruses and these novel rat astroviruses may share a common ancestor.

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

Mimivirus appears most closely related structurally to large algal viruses such as PBCV-1 and other irridoviruses, though it possesses additional features not present in those viruses. The icosahedral capsid consists of 20 large triangular plates joined at their edges to produce the required 12 fivefold vertices. The capsid surface is composed of trimeric major capsid proteins, each subunit of which consists of two jelly roll beta barrels, arranged in a very open hexagonal lattice, with the appearance of a honeycomb. The center-to-center distance of the capsomeres is 14 nm. Because there is some ambiguity as to what lies exactly on the fivefold vertex, there is an uncertainty in the triangulation number T, which could have any one of nine possibilities lying between 972 and 1200.

The fibres that coat particles are reported to have lengths of about 125 nm and are probably anchored to the major capsid proteins or perhaps to an integument layer of protein disposed immediately above the capsid. The most striking and unique feature of mimivirus is a prominent five armed, star-shaped apparatus that occupies one vertex of every virus. While somewhat obscured on the intact virus, it is prominently displayed on particles which lack the coating of fibers. This star shaped assembly, termed a “stargate,” opens up once the virus is inside the host cell to produce a wide opening. The DNA of the virus, which is enclosed inside a membrane sac, then emerges from the interior, fuses with a cellular membrane, and delivers its nucleic acid contents to the cell. Another remarkable is that the DNA does not enter the capsid through the stargate, but through a separate portal, and by a completely different mechanism, in the center of a distal icosahedral face, i.e. at a threefold axis.

Atomic force microscopy investigation of the giant mimivirus. Virology. 2010 404(1): 127-137
Mimivirus was investigated by atomic force microscopy in its native state following serial degradation by lysozyme and bromelain. The 750-nm diameter virus is coated with a forest of glycosylated protein fibers of lengths about 140 nm with diameters 1.4 nm. Fibers are capped with distinctive ellipsoidal protein heads of estimated Mr=25 kDa. The surface fibers are attached to the particle through a layer of protein covering the capsid, which is in turn composed of the major capsid protein (MCP). The latter is organized as an open network of hexagonal rings with central depressions separated by 14 nm. The virion exhibits an elaborate apparatus at a unique vertex, visible as a star shaped depression on native particles, but on defibered virions as five arms of 50 nm width and 250 nm length rising above the capsid by 20 nm. The apparatus is integrated into the capsid and not applied atop the icosahedral lattice. Prior to DNA release, the arms of the star disengage from the virion and it opens by folding back five adjacent triangular faces. A membrane sac containing the DNA emerges from the capsid in preparation for fusion with a membrane of the host cell. Also observed from disrupted virions were masses of distinctive fibers of diameter about 1 nm, and having a 7-nm periodicity. These are probably contained within the capsid along with the DNA bearing sac. The fibers were occasionally observed associated with toroidal protein clusters interpreted as processive enzymes modifying the fibers.

Related:

• Atomic force microscopy
• Marvellous mimivirus
• Mimivirus replicates in the cytoplasm

Some of the most hyperthermophilic bacteria are found within the genus Thermotoga. This article in Microbiology Today explains how these properties could make these micro-organisms promising new sources of biofuels:

Thermotoga first attracted the attention of biotechnologists because of the variety of thermophilic enzymes found in the genus. These bacteria grow on different carbohydrates that are degraded into their sugar monomers before they are taken up and metabolized. It has therefore been possible to isolate, characterize and use a number of highly thermostable hydrolytic and other enzymes from Thermotoga.

Read More

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The major approach to the medical management of HIV infection is the treatment of patients with antiviral drugs. The enzymatic processes of the HIV-1 replication cycle present unique approaches for targeted disruption by pharmacological agents. Due to the high rates of virus production and the mutation rate of the virus, treatment of HIV-1 infection generally includes administration of three agents in combination, referred to as highly active antiretroviral therapy (HAART). Sustained treatment of patients with three active drugs results in suppression of viral replication in peripheral blood to below detection limits of sensitive clinical assays (<50 RNA copies/ml). Continued virologic suppression has led to dramatic increases in the life expectancy of HIV-infected individuals and in time to diagnosis with AIDS, and decreases in HIV-associated morbidity and opportunistic infection. To date, 24 individual drugs have been approved by the United States Food and Drug Administration for the treatment of HIV infection. These drugs are distributed into six major classes:

1. Nucleoside-analog reverse transcriptase inhibitors (NRTI)
2. Non-nucleoside reverse transcriptase inhibitors (NNRTI)
3. Protease inhibitors (PI)
4. Fusion inhibitors
5. Entry Inhibitors – Coreceptor Antagonists
6. Integrase inhibitors

Entry inhibitors represent a new class of antiretroviral agents for the treatment of infection with HIV-1. While resistance to other HIV drug classes has been well described, resistance to this new class is still ill-defined despite considerable clinical use. Several potential mechanisms have been proposed: tropism switching (utilization of CXCR4 instead of CCR5 for entry), increased affinity for the coreceptor, increased rate of virus entry into host cells, and utilization of inhibitor-bound receptor for entry. This review addresses the development of attachment, fusion, and coreceptor entry inhibitors and explores recent studies describing potential mechanisms of resistance.

HIV-1 Entry, Inhibitors, and Resistance. Viruses 2010, 2(5), 1069-1105; doi:10.3390/v2051069

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• Virus-specific HIV drug approved
• More sensitive testing reveals drug-resistant HIV
• Leicester research predicts HIV drug adverse reaction

NK cells have a prominent role in early virus control. Upon activation NK cells control infection either through the lysis of infected cells, or by release of antimicrobial cytokines. This latter function enables them to influence adaptive immune responses as well. NK cells survey their surroundings through numerous inhibitory and activating receptors – integration of these signals determines the activity of an NK cell. More recent data indicate that NK cells may even acquire memory-like function enabling them to respond differently upon recall challenge. The importance of NK cells in control of viral infection is best illustrated by the sheer number of viral evasion mechanisms. These mechanisms include regulation of apoptosis, interference with ADCC, modulation of cytokines and chemokines and function of APCs. Even more numerous are viral techniques dedicated to control of engagement of NK cell receptors. The viruses can downmodulate ligands for activating NK cell receptors, provide competitors and surrogates for cellular ligands, interfere with their translation or target the activating receptors directly.

Despite a remarkable increase in knowledge about relationship between NK cells and viruses there are still many outstanding issues. This review highlights recent progress and current understanding of most important viral immunosubversive mechanisms directed at NK cells with emphasis on receptor-ligand interactions and their impact on overall immunity.

Modulation of natural killer cell activity by viruses. Curr Opin Microbiol. Jun 15 2010

Related:

• Viruses and Apoptosis
• Viruses and Lymphomas

With an estimated annual incidence of over nine million cases, tuberculosis (TB) is believed to be responsible for more adult deaths each year than any other single infectious agent. The highest burden of disease is currently borne by the less developed countries of Africa and Asia where efforts to control TB are hampered by weak health systems and in some settings, by the high prevalence of co-infection with HIV. The recent emergence of multidrugresistant stains that cannot be cured with standard treatments has served to emphasize the urgency of the situation. Control of TB in high burden countries relies on the detection and treatment of infectious cases, most usually by testing patients attending a health clinic that report a cough of at least three weeks duration. The diagnostic tests available in these settings are sputum smear microscopy, an insensitive technique requiring a skilled practitioner and chest radiography, a technique lacking in specificity as well as sensitivity. World Health Organization estimates suggest that in 2006 there were 4 million individuals with undiagnosed tuberculosis. More effective interventions are required to detect and treat infectious cases earlier in the transmission chain, particularly in vulnerable communities with a high prevalence of HIV.

Mycobacterium tuberculosis, the causative agent is spread from person to person via infected aerosols created by patients with respiratory forms of the disease. Bacilli released into the airways following necrosis and destruction of lung tissue may be expelled from the lungs and if released in the form of aerosols may remain airborne and available for inhalation and infection of a new host. Despite being the major mode of transmission there is little data available regarding the exhalation of M. tuberculosis. This paper describes testing of a novel device that utilizes immunosensor and bio-optical technology to detect M. tuberculosis antigen in the breath of humans.

Field test of a novel detection device for Mycobacterium tuberculosis antigen in cough. BMC Infectious Diseases 2010, 10: 161 doi:10.1186/1471-2334-10-161
Tuberculosis is a highly infectious disease that is spread from person to person by infected aerosols emitted by patients with respiratory forms of the disease. We describe a novel device that utilizes immunosensor and bio-optical technology to detect M. tuberculosis antigen (Ag85B) in cough and demonstrate its use under field conditions during a pilot study in an area of high TB incidence.
The TB Breathalyzer device (Rapid Biosensor Systems Ltd) was field tested in the outpatient clinic of Adama Hospital, Ethiopia. Adults seeking diagnosis for respiratory complaints were tested. Following nebulization with 0.9% saline patients were asked to cough into a disposable collection device where cough aerosols were deposited. Devices were then inserted into a portable instrument to assess whether antigen was present in the sample. Demographic and clinical data were recorded and all patients were subjected to chest radiogram and examination of sputum by Ziehl-Nielsen microscopy. In the absence of culture treatment decisions were based on smear microscopy, chest x-ray and clinical assessment. Breathalyzer testing was undertaken by a separate physician to triage and diagnostic assessment.
Sixty individuals were each subjected to a breathalyzer test. The procedure was well tolerated and for each patient the testing was completed in less than 10 min. Positive breath test results were recorded for 29 (48%) patients. Of 31 patients with a diagnosis of tuberculosis 23 (74%) were found positive for antigen in their breath and 20 (64%) were smear positive for acid fast bacilli in their sputum. Six patients provided apparent false positive breathalyzer results that did not correlate with a diagnosis of tuberculosis.
We propose that the breathalyzer device described warrants further investigation as a tool for studying exhalation of M. tuberculosis. The portability, simplicity of use and speed of the test device suggest it may also find use as a tool to aid early identification of infectious cases. We recommend studies be undertaken to determine the diagnostic sensitivity and specificity of the device when compared to microbiological and clinical indicators of tuberculosis disease.

Related:

• Why is TB more common in men than in women?
• Origin and spread of Mycobacterium tuberculosis
• Public health and bovine tuberculosis

Plasmodium falciparum is a blood parasite that lives for the most part inside red cells. It is responsible for the death of 1-2 million people through malaria every year. The mechanisms by which the parasite invades red cells are complex and not completely understood. For many years it has been known that proteins called glycophorins are used by the parasite to gain entry into the red cell. However, the existence of another protein that allows entry independent of glycophorins has been suspected for nearly as long. The identity of the alternative protein has been a difficult mystery to solve.

Researchers have now identified an alternative protein used by P. falciparum to invade red blood cells. The results may aid the development of a future vaccine for malaria. The researchers identified complement receptor 1 as the protein that enables P. falciparum to invade red blood cells. Proteins called glycophorins are used by the parasite to gain entry into the red cell. However, because infection can take place without glycophorins, researchers suspected that another protein is also involved. Complement receptor 1 (CR1), also known to help protect red cells from attack by the immune system, has been suspected of having other roles in the development of malaria complications. The team was able to demonstrate that this protein is important in the invasion of red cells by using several laboratory strains of malaria as well as strains obtained from Kenya.

P. falciparum may use the CR1 protein instead of glycophorins if the parasite encounters a variant that lacks the glycophorin receptor; if the immune system mounts a response against parasite proteins involved in the dominant pathway due to a previous infection; or if the host were treated with a vaccine that blocks the glycophorin pathway. The recognition of the additional role of complement receptor 1 in red cell invasion will allow the definitive identification of malaria proteins that interact with it and that could be used in a future vaccine cocktail to block red cell invasion.

Complement Receptor 1 Is a Sialic Acid-Independent Erythrocyte Receptor of Plasmodium falciparum. 2010 PLoS Pathog 6(6): e1000968. doi:10.1371/journal.ppat.1000968
Plasmodium falciparum is a highly lethal malaria parasite of humans. A major portion of its life cycle is dedicated to invading and multiplying inside erythrocytes. The molecular mechanisms of erythrocyte invasion are incompletely understood. P. falciparum depends heavily on sialic acid present on glycophorins to invade erythrocytes. However, a significant proportion of laboratory and field isolates are also able to invade erythrocytes in a sialic acid-independent manner. The identity of the erythrocyte sialic acid-independent receptor has been a mystery for decades. We report here that the complement receptor 1 (CR1) is a sialic acid-independent receptor for the invasion of erythrocytes by P. falciparum. We show that soluble CR1 (sCR1) as well as polyclonal and monoclonal antibodies against CR1 inhibit sialic acid-independent invasion in a variety of laboratory strains and wild isolates, and that merozoites interact directly with CR1 on the erythrocyte surface and with sCR1- coated microspheres. Also, the invasion of neuraminidase-treated erythrocytes correlates with the level of CR1 expression. Finally, both sialic acid-independent and dependent strains invade CR1 transgenic mouse erythrocytes preferentially over wild-type erythrocytes but invasion by the latter is more sensitive to neuraminidase. These results suggest that both sialic acid-dependent and independent strains interact with CR1 in the normal red cell during the invasion process. However, only sialic acid-independent strains can do so without the presence of glycophorin sialic acid. Our results close a longstanding and important gap in the understanding of the mechanism of erythrocyte invasion by P. falciparum that will eventually make possible the development of an effective blood stage vaccine.

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• New ways to beat malaria
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