MicrobiologyBytes

BBC: veruccas and warts

Most bacteria contain in their envelope a peptidoglycan (or “murein”) layer which protects the cell from rupture as a result of osmotic pressure (turgor) of the cytoplasm and which maintains the specific shape (sphere, rod, spiral or other) of the bacterial cell. Although the chemical composition varies in different species, peptidoglycan is always made of glycan strands that are crosslinked by short peptides. Characteristic structural features include N-acetylmuramic acid (MurNAc), d-amino acids and unusual amide bonds. Many important questions on the structure and growth of the sacculus remain unanswered. Of particular interest is the architecture of peptidoglycan, the knowledge of which is needed to understand the mechanism(s) of enlargement of the sacculus in growing and dividing cells.

A crucial feature of the architecture of peptidoglycan is the orientation of the glycan strands and peptides with regard to surface and axis of the cell. However, the large size, structural heterogeneity and flexibility of the peptidoglycan sacculi preclude crystal formation and hence determination of the crystal structure. Furthermore, the glycan strands and peptides cannot be seen by conventional electron microscopy because their dimensions are of the same scale as the inevitable staining and fixation artifacts. Thus, current models of the architecture are based on chemical and biophysical data which, however, are limited to just a few species. Two mutually exclusive models are currently being discussed: the classical “layered” model in which both the glycan strands and peptide crosslinks run parallel (horizontal) to the cytoplasmic membrane, and the “scaffold” model in which the glycan strands run perpendicular to the membrane and the peptide crosslinks run parallel to the membrane. The existence of these fundamentally different models and the arguments about them have had one very positive effect: structural biologists are beginning to regain their interest in this problem utilizing current technologies and new methods.

In 2008, two important articles on the architecture of peptidoglycan were published. The first describes the architecture of sacculi isolated from Gram-negative species analyzed by electron cryotomography. The second reports on the architecture of peptidoglycan isolated from the Gram-positive, rod-shaped Bacillus subtilis as observed by atomic force microscopy (AFM). Although the new data support a layered architecture in Gram-negative bacteria, the situation appears to be more complex in B. subtilis. The data suggest that neither of the two models apply. This article summarizes the state of knowledge of peptidoglycan, discusses current models for Gram-negative and Gram-positive bacteria and notes the many unanswered questions about the architecture of peptidoglycan which remain.

Architecture of peptidoglycan: more data and more models. Trends in Microbiology, 8 January 2010. doi:10.1016/j.tim.2009.12.004

Related:

• Peptidoglycan – the strength and weakness of bacteria
• Getting bacteria into shape

Trypanosoma brucei and related subspecies are protozoan parasites that cause African trypanosomiasis in humans and animals, a deadly disease with devastating health and economic consequences. The T. brucei flagellum and flagellar motility are central to disease pathogenesis in the mammalian host and parasite development in the tsetse fly vector. Recent functional studies have revealed that the T. brucei flagellum is an essential and multifunctional organelle with critical roles in motility, cellular morphogenesis, cell division, immune evasion, and potentially, sensory perception. Concurrently, genomic and proteomic studies have significantly expanded the inventory of known trypanosome flagellar proteins. Because the flagellum is an essential organelle, understanding unique aspects of the T. brucei flagellum may uncover novel drug targets. In addition, T. brucei is emerging as a powerful experimental system for studies of conserved aspects of flagellum/cilium biology (flagellum and cilium are interchangeable terms for the same organelle), with direct relevance to other eukaryotes, including humans, in which flagellum/cilium defects underlie many heritable, fatal, and debilitating diseases.

African trypanosomes are devastating human and animal pathogens. Trypanosoma brucei rhodesiense and T. b. gambiense subspecies cause the fatal human disease known as African sleeping sickness. It is estimated that several hundred thousand new infections occur annually and the disease is fatal if untreated. T. brucei is transmitted by the tsetse fly and alternates between bloodstream-form and insect-form life cycle stages that are adapted to survive in the mammalian host and the insect vector, respectively. The importance of the flagellum for parasite motility and attachment to the tsetse fly salivary gland epithelium has been appreciated for many years. Recent studies have revealed both conserved and novel features of T. brucei flagellum structure and composition, as well as surprising new functions that are outlined in this review. These discoveries are important from the standpoint of understanding trypanosome biology and identifying novel drug targets, as well as for advancing our understanding of fundamental aspects of eukaryotic flagellum structure and function.

The Trypanosoma brucei flagellum: moving parasites in new directions. Ann Rev Microbiol. 2009 63: 335-362

Related:

• Antigenic Variation in African Trypanosomes
• Trypanosomes and immune evasion
• BILBO and the trypanosomes

Endospores are unique among bacterial spores in that they are produced inside of another cell (the mother cell) and, upon maturation, are released as free spores by lysis of the mother cell. They are readily recognized under phase-contrast microscopy by their phase bright (refractile) appearance. They also exhibit diagnostic features under electron microscopy, such as a protein shell consisting of an inner coat and an electron dense, outer coat. Endospores are composed of numerous molecules found, thus far, only in bacterial endospores. These molecules include most of the proteins that encase the spore in a protective shell (called the coat), a family of DNA-protective proteins known as SASP that are bound to the chromosome, and a unique small molecule, dipicolinic acid. All previously known examples of endospore-forming bacteria are members of the low G+C group of Gram-positive bacteria (Firmicutes) belonging either to Bacilli or to Clostridia, and in all cases in which a genome sequence is available, orthologs of genes involved in endospore formation are readily seen. The Mycobacterium genus is a member of the high G+C group of Gram-positive bacteria (Actinobacteria) for which there are no prior claims of endospore formation. Certain members of the group, such as Streptomyces, do produce spores, but spores of a fundamentally different kind that are not produced inside a mother cell.

A recent publication in PNAS reported that M. marinum and M. bovis bacillus Calmette–Guérin produce a type of spore known as an endospore, which had been observed only in the low G+C group of Gram-positive bacteria. Evidence was presented that the spores were similar to endospores in ultrastructure, in heat resistance and in the presence of dipicolinic acid. This paper reports that the genomes of Mycobacterium species and those of other high G+C Gram-positive bacteria lack orthologs of many, if not all, highly conserved genes diagnostic of endospore formation in the genomes of low G+C Gram-positive bacteria. It also failed to detect the presence of endospores by light microscopy or by testing for heat-resistant colony-forming units in aged cultures of M. marinum. Finally, we failed to recover heat-resistant colony-forming units from frogs chronically infected with M. marinum. It concludes that it is unlikely that Mycobacterium is capable of endospore formation.

Do mycobacteria produce endospores? PNAS USA December 22 2009 doi: 10.1073/pnas.0911299107

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• Anthrax and other spore-forming bacteria
• Why is TB more common in men than in women?
• Foamy macrophages allow TB agent to survive in infected individuals

All viruses need to get into host cells, and eventually, get out of them again. Because of the cell wall, plant viruses face particular problems getting into cells, but we’ll think about them another time. To get into cells, most animal viruses use endocytosis, the process by which cells absorb molecules (such as proteins) from outside. Which brings us to clathrin, shown in this excellent video:

Budding or exocytosis of virus particles (at least, the ones which don’t cheat by causing lysis of infected cells) also involves the cytoskeleton, all of which make clathrin a pretty important molecule for animal viruses.

Related:

• Dissecting the Cell Entry Pathway of Dengue Virus
• Imaging Poliovirus Entry in Live Cells
• How can lytic viruses be evolutionarily competitive?

The malaria parasite life cycle constitutes one of the most complicated and fascinating life cycles of any organism and thus poses intriguing areas of study for cell biology, molecular biology, and immunology alike. Malaria as a disease is devastating developing countries, especially those in sub-Saharan Africa, causing approximately one million deaths each year, which are mainly attributable to a single parasite species, Plasmodium falciparum. The intricacy of malaria parasite biology has vexed vaccinologists and immunologists for nearly a century and is a major impediment to the development of a fully protective vaccine. A major part of the complexity associated with the malaria parasite life cycle is due to the parasite’s ability to change its cellular and molecular makeup, which is controlled by a genome with more than 5000 recognized genes, and develop in intracellular and extracellular niches in the mammalian host and the mosquito vector.

Plasmodium sporozoites are the product of a complex developmental process in the mosquito vector and are destined to infect the mammalian liver. Attention has been drawn to the mosquito stages and pre-erythrocytic stages owing to recognition that these are bottlenecks in the parasite life cycle and that intervention at these stages can block transmission and prevent infection. Parasite progression in the Anopheles mosquito, sporozoite transmission to the mammalian host by mosquito bite, and subsequent infection of the liver are characterized by extensive migration of invasive stages, cell invasion, and developmental changes. Preparation for the liver phase in the mammalian host begins in the mosquito with an extensive reprogramming of the sporozoite to support efficient infection and survival. This review discusses what is known about the molecular and cellular basis of the developmental progression of parasites and their interactions with host tissues in the mosquito and during the early phase of mammalian infection.

Malaria parasite development in the mosquito and infection of the mammalian host. Ann Rev Microbiol. 2009 63: 195-221

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• The origin of malignant malaria
• Nature Collections – Malaria
• New ways to beat malaria

In 1518, one of the strangest epidemics in recorded history struck the city of Strasbourg. Hundreds of people were seized by an irresistible urge to dance, hop and leap into the air. In houses, halls and public spaces, as fear paralyzed the city and the members of the elite despaired, the dancing continued with mindless intensity. Seldom pausing to eat, drink or rest, many of them danced for days or even weeks. And before long, the chronicles agree, dozens were dying from exhaustion. What was it that could have impelled as many as 400 people to dance, in some cases to death?

Medieval dancing epidemics were not unrelated events: they were linked both in time and space. Every one of the ten or so outbreaks between the late 1300s and 1518 happened along the Rhine and Mosel rivers. In 1374, for instance, the crazed dance gradually spread out from an epicentre around Aachen, Liege and Maastricht to neighbouring towns such as Ghent, Utrecht, Metz, Trier and, eventually, Strasbourg. Moreover, outbreaks of compulsive dancing virtually always struck in or close to places affected by earlier outbreaks. Maastricht, Trier, Zurich and Strasbourg each experienced two or more episodes. There are also several reports of compulsive dancing after 1518. All of these, crucially, took place close to the Rhine, and all but one within a short ride of Strasbourg itself.

How can we explain this striking epidemiological picture? One suggestion is that wild dancing formed part of the ecstatic ritual of a heretical sect, an energetic counterpart of the flagellant’s cult. There are two main difficulties with this theory. First, in lucid moments the dancers implored bystanders and priests to come to their aid. There is absolutely no evidence that the dancers wanted to dance. On the contrary, they expressed fear and desperation. Second, the authorities consistently saw the afflicted not as heretics but as the victims of diabolical possession or divine curse, and treated them accordingly. The dancers were subject to exorcisms or sent on pilgrimages. Never were they hauled before the inquisition.

Other authors have sought a chemical or biological origin for the dancing mania, and the chief contender has been ergot, a mould that grows on the stalks of damp rye. While seductively simple, this hypothesis is untenable. The chemicals contained in ergot do not allow for sustained dancing. They can certainly trigger violent convulsions and delusions, but not coordinated movements that last for days. Yet while the dancers were free from ergot, they almost certainly were delirious. Only in an altered state of consciousness could they have tolerated such extreme fatigue and the searing pain of sore, swollen and bleeding feet. Moreover, witnesses consistently spoke of the victims as being entranced, seeing terrifying visions and behaving with wild, crazy abandon. So what could have plunged hundreds of people into trances so deep that remorseless dancing became possible? Psychologists, neurologists and anthropologists have identified severe psychological distress as a factor increasing the likelihood of an individual entering an altered state. It is unlikely to be a coincidence, therefore, that in the year 1518 many people in Strasbourg were experiencing truly exceptional levels of hunger and mental anguish.

In a spin: the mysterious dancing epidemic of 1518. Endeavour. 2008 32(3): 117-121. doi: 10.1016/j.endeavour.2008.05.001

Marine dinoflagellates produce a diverse suite of complex toxins, yet the biological reason remains unclear. Among this group, Karlodinium veneficum is a small (∼8–12 μm) phytoplankton, common in coastal aquatic ecosystems. It has a mixed nutritional mode, relying on both photosynthesis and phagotrophy for growth (mixotrophy). It is frequently present in relatively low cell abundance, but is capable of forming intense blooms that have been associated with fish kills. Karlotoxins (KmTxs), which are produced by K. veneficum, generate pores in membranes with sterols and increase the ionic permeability of cell membranes, resulting in membrane depolarization, disruption of motor functions, osmotic cell swelling, and lysis. These measured effects have been based on purified toxins. Karlotoxin type and cell amount vary with K. veneficum strain, culture conditions, and geographic location. Along the U.S. East Coast, K. veneficum strains from south of the Chesapeake Bay produce karlotoxin 2 (KmTx- 2), whereas those from within the Bay produce karlotoxin 1 (KmTx-1).

To date, the perceived ecological role for toxins has been relief from grazing pressures. New research suggests that karlotoxins also serve as a predation instrument. Using high-speed holographic microscopy, researchers measured the swimming behavior of several toxic and nontoxic strains of K. veneficum and their prey, Storeatula major, within dense suspensions. The selected strains produce toxins with varying potency and dosages, including a nontoxic one. Results clearly show that mixing the prey with the predatory, toxic strains causes prey immobilization at rates that are consistent with the karlotoxins’ potency and dosage. Even prey cells that continue swimming slow down after exposure to toxic predators. The swimming characteristics of predators vary substantially in pure suspensions, as quantified by their velocity, radii of helical trajectories, and direction of helical rotation. When mixed with prey, all toxic strains that are involved in predation slow down. Furthermore, they substantially reduced their predominantly vertical migration, presumably to remain in the vicinity of their prey. Conversely, the nontoxic control strain does not alter its swimming and does not affect prey behavior. In separate experiments, the authors show that exposing prey to exogenous toxins also causes prey immobilization at rates consistent with potency. Clearly, the toxic predatory strains use karlotoxins as a means of stunning their prey, before ingesting it. These findings add to our understanding of why some dinoflagellates produce such complex toxin molecules.

A dinoflagellate exploits toxins to immobilize prey prior to ingestion. PNAS USA January 19 2010. doi: 10.1073/pnas.0912254107

The Shiga toxin family, a group of structurally and functionally related exotoxins, includes Shiga toxin from Shigella dysenteriae serotype 1 and the Shiga toxins that are produced by enterohaemorrhagic Escherichia coli (EHEC) strains. The existence of different, interchangeable terms to describe very similar toxins has historical reasons. The Japanese microbiologist Kiyoshi Shiga was the first to characterize the bacterial origin of dysentery caused by S. dysenteriae, in 1897. In 1977, Konowalchuk discovered a group of E. coli isolates that produced a factor that was able to kill Vero cells in culture. The factor was termed verotoxin, and the bacteria were termed verotoxin-producing E. coli (VTEC). O’Brien and colleagues recognized in the early 1980s that some E. coli isolates produced a toxin that was related to Shiga toxin and named these organisms Shiga-like toxin-producing E. coli (STEC). In 1983, it was recognized that STEC strains are associated with haemolytic uraemic syndrome (HUS). Researchers eventually realized that they were studying identical or highly related toxins.

Shiga toxin is the prototype of the Shiga toxin family and nearly identical to the E. coli-produced Shiga toxin 1 (Stx1), differing by a single amino acid. Severe disease has been epidemiologically linked to the presence of Stx2. Although Stx1 and Stx2 share a common receptor and possess the same intracellular mechanism of action, they are immunologically distinct and only 56% identical at the amino acid sequence level.

These toxins have received considerable attention not only from microbiologists but also in the field of cell biology, where it has become a powerful tool to study intracellular trafficking. In this Review, we summarize the Shiga toxin family members and their structures, receptors, trafficking pathways and cellular targets. This review discusses how Shiga toxin affects cells not only by inhibiting protein biosynthesis but also through the induction of signalling cascades that lead to apoptosis. It also discusses how Shiga toxins might be exploited in cancer therapy and immunotherapy.

Shiga toxins – from cell biology to biomedical applications. 2010 Nature Reviews Microbiology 8: 105-116. doi: 10.1038/nrmicro2279

Related:

• Bacterial toxins
• The continuing evolution of E. coli O157:H7
• Botox is bonkers