MicrobiologyBytes

Apicomplexans are an important group of pathogens that include the causative agents of malaria, toxoplasmosis, and cryptosporidiosis. These single-celled eukaryotic parasites evolved from photosynthetic algae. A remnant chloroplast, called the apicoplast, is a hold-over from this more benign past in the ocean. The apicoplast is essential for parasite growth and development and therefore a potential target for drug therapy. The fact that humans and animals lack chloroplasts suggests that using approaches to target the apicoplast may provide parasite specificity. What are the critical functions of the apicoplast that should be targeted? In addition to the obvious medical relevance this question has broader biological implications. Why do organisms maintain an ancient symbiotic relationship when the initial rationale for this relationship has fallen by the evolutionary wayside?

A new study provides important clues. It demonstrates that antibiotic-induced loss of the apicoplast in cultured malaria parasites can be chemically rescued by providing isopentenyl-pyrophosphate (IPP) in the medium. IPP is generated by the apicoplast resident isoprenoid biosynthesis pathway and is apparently the one apicoplast metabolite that the parasite cannot live without in the red blood cell. This finding could be of great importance for the development of drugs and vaccines. The ability to produce and maintain parasite lines that lack the apicoplast also offers exciting experimental possibilities for the future.

What Do Human Parasites Do with a Chloroplast Anyway? (2011) PLoS Biol 9(8): e1001137. doi:10.1371/journal.pbio.1001137

Tweet

A number of flaviviruses constitute a significant threat to global health. Dengue virus (DENV) infection causes around 21,000 human deaths annually, and it is estimated that at least 120 countries have endemic DENV transmission, whilst in recent years, West Nile virus (WNV) has become more prominent as a zoonotic agent, particularly in North America where the virus first emerged in 1999 and rapidly spread across the continent. WNV has now emerged in a number of European countries, particularly around the Mediterranean basin, where infections in humans, horses and birds have been reported.

Cross-reactivity of sera raised against one flavivirus recognising another flavivirus has been well documented. One consequence of flavivirus cross-reactivity is the occurrence of false-positive results, yet cross-reactivity can lead to cross-protection. Understanding and manipulating the cross-reactive properties of flaviviruses has the potential to assist the development of effective broad-spectrum human vaccines against WNV and other existing and emerging flaviviruses.

Flavivirus-induced antibody cross-reactivity. J Gen Virol. Sep 7 2011
Dengue viruses (DENV) cause countless human deaths each year, whilst West Nile virus (WNV) has re-emerged as an important human pathogen. There are currently no WNV or DENV vaccines licensed for human use, yet vaccines exist against other flaviviruses. To investigate flavivirus cross-reactivity, sera from a human cohort with a history of vaccination against tick-borne encephalitis virus (TBEV), Japanese encephalitis virus (JEV) and yellow fever virus (YFV) were tested for antibodies by plaque reduction neutralisation test. Neutralisation of Louping ill virus (LIV) occurred, but no significant neutralisation of Murray Valley encephalitis virus (MVEV) was observed. Sera from some individuals vaccinated against TBEV and JEV neutralised WNV, which was enhanced by YFV vaccination in some recipients. Similarly, some individuals neutralised DENV-2, but this was not significantly influenced by YFV vaccination. Antigenic cartography techniques were used to generate a geometric illustration of the neutralisation titres of selected sera against WNV, TBEV, JEV, LIV, YFV and DENV-2. This demonstrated the individual variation in antibody responses. Most sera had detectable titres against LIV and some had titres against WNV and DENV-2. Generally, LIV titres were similar to titres against TBEV, confirming the close antigenic relationship between TBEV and LIV. JEV was also antigenically closer to TBEV than WNV, using these sera. The use of sera from individuals vaccinated against multiple pathogens is unique relative to previous applications of antigenic cartography techniques. It is evident from these data that notable differences exists between amino acid sequence identity and mapped antigenic relationships within the family Flaviviridae.

Tweet

Humans have suffered from the burden of malarial infections for thousands of years, and the disease has greatly influenced human evolution and history. Malaria remains a devastating disease, and in developing countries within Africa, South America, and Asia, the size of its burden has stifled economic growth and development. Despite successful eradication campaigns in North America and Europe, global cases of the disease show little decline, and current improvements rely on pyrethroid treated bed nets and combination therapeutics containing artemisinin derivatives, both of which are susceptible to emerging resistance. Our ability to counter these vulnerabilities with new agents is hampered by the modest number of fully validated drug targets and our limited understanding of many aspects of parasite biology.

The Next Opportunity in Anti-Malaria Drug Discovery: The Liver Stage. (2011) PLoS Pathog 7(9): e1002178. doi:10.1371/journal.ppat.1002178
Malaria afflicts 350–500 million people annually, and this debilitating and deadly infectious disease exacts a heavy toll on susceptible populations around the globe. Efforts to find effective, safe, and low-cost drugs for malaria have sharply increased in recent years. Almost all of these efforts have focused on the cyclic blood stage of the disease, partly because the parasites can be easily maintained in culture through addition of human red blood cells to the growth medium, and partly because blood stage infection causes malaria’s characteristic symptoms. However, the asymptomatic liver stage, which the parasite goes through only once in its life history, presents the best opportunity for developing drugs that both hit new targets and also could be used in highly desirable eradication campaigns. Recent research, especially on the frequency of differentially expressed genes in blood and liver stage parasites, supports the feasibility of discovering stage-specific drugs. Discovering these drugs will require a high-throughput liver stage phenotypic screen comparable to the existing blood stage screens, and the basic tools for such a screen have recently been created.

Tweet

Stomach ulcers are caused by chronic infection with the bacterium Helicobacter pylori, which is also the leading risk factor for stomach cancer. One reason for the cancer risk could be that the pathogen creates breaks in the DNA molecules of infected cells:

Carcinogenic bacterial pathogen Helicobacter pylori triggers DNA double-strand breaks and a DNA damage response in its host cells. PNAS USA 108: 14944–14949 (2011)
The bacterial pathogen Helicobacter pylori chronically infects the human gastric mucosa and is the leading risk factor for the development of gastric cancer. The molecular mechanisms of H. pylori-associated gastric carcinogenesis remain ill defined. In this study, we examined the possibility that H. pylori directly compromises the genomic integrity of its host cells. We provide evidence that the infection introduces DNA double-strand breaks (DSBs) in primary and transformed murine and human epithelial and mesenchymal cells. The induction of DSBs depends on the direct contact of live bacteria with mammalian cells. The infection-associated DNA damage is evident upon separation of nuclear DNA by pulse field gel electrophoresis and by high-magnification microscopy of metaphase chromosomes. Bacterial adhesion (e.g., via blood group antigen-binding adhesin) is required to induce DSBs; in contrast, the H. pylori virulence factors vacuolating cytotoxin A, γ-glutamyl transpeptidase, and the cytotoxin-associated gene (Cag) pathogenicity island are dispensable for DSB induction. The DNA discontinuities trigger a damage-signaling and repair response involving the sequential ataxia telangiectasia mutated (ATM)-dependent recruitment of repair factors—p53-binding protein (53BP1) and mediator of DNA damage checkpoint protein 1 (MDC1)—and histone H2A variant X (H2AX) phosphorylation. Although most breaks are repaired efficiently upon termination of the infection, we observe that prolonged active infection leads to saturation of cellular repair capabilities. In summary, we conclude that DNA damage followed by potentially imprecise repair is consistent with the carcinogenic properties of H. pylori and with its mutagenic properties in vitro and in vivo and may contribute to the genetic instability and frequent chromosomal aberrations that are a hallmark of gastric cancer.

Tweet

A hallmark of negative-strand RNA viruses is that their genomes never exist as free RNA, but instead are always assembled with many copies of a single nucleoprotein, N, to form highly stable nucleocapsids. Moreover, viral genomes are the only RNAs in infected cells that are assembled with N. The mechanism by which this specific association occurs, for both the segmented and non-segmented viruses, has recently become clearer due to our expanding knowledge of N protein and nucleocapsid structures.

Nucleoproteins and nucleocapsids of negative-strand RNA viruses. Curr Opin Microbiol. Aug 6 2011

Tweet

One of the great advances in medical technology has unwittingly spawned a serious threat to public health. Implanted medical devices, from cardiac stents to artificial hip joints, are commonly infected with biofilms, complex microbial communities that can prove remarkably resistant to host defenses and treatment. It appears, however, that biofilms, even those arising from the same microbe species, may harbor innate differences in their response to treatments like antifungal agents. Understanding how these microbe colonies might produce structures that appear similar but have very different physical properties and functions is a critical step in figuring out how to overcome antimicrobial resistance.

In humans, Candida albicans can cause problems like oral thrush and yeast infections. Far more serious is its increasing tendency to colonize catheters, heart valves, and other medical devices, where it serves as a seeding source for potentially deadly bloodstream infections. The finding that C. albicans can form two different types of biofilm is interesting not only because it is an example of how similar structures can have very different functions, but also because it provides information about how signaling pathways may evolve by modifying preexisting signaling modules for entirely new purposes. This phenomenon may be widespread, extending beyond slimy fungal biofilms to a variety of organisms. On a practical note, when researchers are designing new methods to discourage fungal biofilms it will be useful for them to keep in mind that there are two types of biofilms being formed by C. albicans.

A Tale of Two Biofilms. 2011 PLoS Biol 9(8): e1001119. doi:10.1371/journal.pbio.1001119

Tweet

Human bocavirus (HBoV) was discovered in 2005. HBoV has been detected in patients suffering from respiratory infections and gastrointestinal diseases, but a proof that HBoV is the causative agent in such cases is missing as it remains impossible so far to fulfil Koch’s modified postulates. The latter problem is caused by the fact that HBoV is difficult to propagate in cell culture and that no animal model is available.

Since HBoV infections are accompanied by co-pathogens in a very high frequency, it has been suggested that HBoV is a passenger rather than a pathogen in airway infections, despite the fact that HBoV causes a productive infection with viral shedding, viremia, and putative persistence in different organs. Although HBoV meanwhile was classified as an autonomous parvovirus rather than a Dependovirus like the Adeno-associated virus (AAV), there remains the possibility that HBoV infections depend on helper viruses or at least contributes synergistically to the clinical course of disease.

Does human bocavirus infection depend on helper viruses? A challenging case report. Virology Journal 2011, 8: 417 doi:10.1186/1743-422X-8-417
A case of severe diarrhoea associated with synergistic human bocavirus type 1 (HBoV) and human herpes virus type 6 (HHV6) is reported. The case supports the hypotheses that HBoV infection under clinical conditions may depend on helper viruses, or that HBoV replicates by a mechanism that is atypical for parvoviruses, or that HBoV infection can be specifically treated with cidofovir.

Tweet

Fifteen years ago, in a series of elegant studies, Hackstadt and colleagues showed that the obligate intracellular bacteria Chlamydia trachomatis save on their lipid needs by incorporating sphingomyelins (SMs) made by their host. Shortly after, Hatch and McClarty’s teams reported that several eukaryotic glycerophospholipids are also trafficked from the host to the bacteria, which replace host-synthesized straight-chain fatty acids by their own branched-chain fatty acids. Even cholesterol, a lipid rarely found in bacteria, was shown to accumulate in Chlamydia. As a result of this intense exploitation of host lipids, the composition of the bacterial membrane is closer to that of a eukaryotic cell than to that of a prokaryote.

Throughout their developmental cycle, chlamydiae reside within a membrane-bounded compartment, the inclusion. How they acquire host lipids remains an open question. Possible mechanisms studied so far involve vesicular trafficking from host compartments, including vesicular traffic out of the Golgi apparatus, fusion with multivesicular body–derived vesicles, and engulfment of lipid droplets.

Rerouting of Host Lipids by Bacteria: Are You CERTain You Need a Vesicle? (2011) PLoS Pathog 7(9): e1002208. doi:10.1371/journal.ppat.1002208

Tweet

Research in virology is driven towards the characterization of a limited number of socioeconomically important pathogens, mostly those infecting humans. Yet, characterization of other viruses may advance our understanding of these topical pathogens and the fundamentals of virology. A recent paper describes the discovery of a virus of unknown clinical relevance that has many remarkable features. The virus was called Nam Dinh virus (NDiV) after a Vietnamese province. It is a mosquito-borne virus with a 20.2 kilobase genome, the largest among non-segmented single-stranded RNA viruses of insects. Employing bioinformatics tools, the authors show that NDiV prototypes a new family and is a missing evolutionary link connecting the distantly related nidoviruses with small and large genomes, including important and diverse pathogens such as porcine respiratory and reproductive syndrome virus (~15-kilobase genome) and SARS coronavirus (~30 kilobases), respectively. NDiV and large nidoviruses form a phylogenetic cluster and share a set of core replicative enzymes. They exclusively encode an exoribonuclease that presumably controls replication fidelity. Its acquisition may have promoted the emergence of viruses with single-stranded RNA genomes larger than ~20 kilobases. This study highlights the benefits of broad virus discovery efforts for fundamental and applied research.

Discovery of the First Insect Nidovirus, a Missing Evolutionary Link in the Emergence of the Largest RNA Virus Genomes. (2011) PLoS Pathog 7(9): e1002215. doi:10.1371/journal.ppat.1002215
Nidoviruses with large genomes (26.3–31.7 kb; ‘large nidoviruses’), including Coronaviridae and Roniviridae, are the most complex positive-sense single-stranded RNA (ssRNA+) viruses. Based on genome size, they are far separated from all other ssRNA+ viruses (below 19.6 kb), including the distantly related Arteriviridae (12.7–15.7 kb; ‘small nidoviruses’). Exceptionally for ssRNA+ viruses, large nidoviruses encode a 3′-5′exoribonuclease (ExoN) that was implicated in controlling RNA replication fidelity. Its acquisition may have given rise to the ancestor of large nidoviruses, a hypothesis for which we here provide evolutionary support using comparative genomics involving the newly discovered first insect-borne nidovirus. This Nam Dinh virus (NDiV), named after a Vietnamese province, was isolated from mosquitoes and is yet to be linked to any pathology. The genome of this enveloped 60–80 nm virus is 20,192 nt and has a nidovirus-like polycistronic organization including two large, partially overlapping open reading frames (ORF) 1a and 1b followed by several smaller 3′-proximal ORFs. Peptide sequencing assigned three virion proteins to ORFs 2a, 2b, and 3, which are expressed from two 3′-coterminal subgenomic RNAs. The NDiV ORF1a/ORF1b frameshifting signal and various replicative proteins were tentatively mapped to canonical positions in the nidovirus genome. They include six nidovirus-wide conserved replicase domains, as well as the ExoN and 2′-O-methyltransferase that are specific to large nidoviruses. NDiV ORF1b also encodes a putative N7-methyltransferase, identified in a subset of large nidoviruses, but not the uridylate-specific endonuclease that – in deviation from the current paradigm – is present exclusively in the currently known vertebrate nidoviruses. Rooted phylogenetic inference by Bayesian and Maximum Likelihood methods indicates that NDiV clusters with roniviruses and that its branch diverged from large nidoviruses early after they split from small nidoviruses. Together these characteristics identify NDiV as the prototype of a new nidovirus family and a missing link in the transition from small to large nidoviruses.

Tweet