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when S. cerevisiae is injected into laboratory mammals. Chitin is a /3(l-4-)-linked

polymer of jV-acetylglucosamine. It confers enormous mechanical strength. In S.

cerevisiae a ring of chitin is formed at the mother-bud junction (see Fig. 2.2). This ring

persists after cell separation and is referred to as the 'bud scar'. Chitin is readily stained

with the optical brightener Calcofluor White, and all of the bud scars on a cell can

easily be visualized. Thus, it is possible to determine both the 'age' of a cell (by counting

the number of bud scars) and the ploidy of the cell (by observing the pattern of bud

scars, because, as explained earlier, haploids and diploids have different patterns of

bud formation). Indeed, ageing research is also possible in this organism (Kennedy &

Guarente 1996). In spore walls, the outermost layers contain a special polymer which

is based upon dityrosine (Briza et al. 1990).

Candida albicans

Pharmaceutical and clinical significance

Candida albicans is a dimorphic organism which is part of the normal body flora of

humans. For the majority of normal healthy individuals it will never cause any problems.

However, in a number of settings it can cause severe disruption to lifestyle or even

death. In the USA it is now the third most frequent cause of nosocomial (hospital

acquired) infections. Post-operative infection arises typically where a patient has been

in intensive care for weeks and has undergone several cycles of bacterial infections

and high dose antibacterial therapy. In excess of 50% of deep-seated Candida infections

are lethal. Persons suffering from AIDS, transplant patients and other immuno-

compromised individuals are at even greater risk. The azole family of antifungal

compounds (see Chapter 5) is frequently deployed but several of these block the

metabolism of cyclosporins (which are administered for chronic immunosuppressive

therapy) and thereby increase immunosupressivity. A possible alternative antifungal

drug is amphotericin B, but this interacts with cyclosporins to give increased nephro-

toxicity. Catheterized patients can become infected with C. parapsilosis which causes

problems by virtue of its ability to form biofilm. In many apparently normal women,

C. albicans causes vaginal thrush which can be so extreme as to incapacitate. Some

denture-wearers and malnourished children can develop thrush in the mouth; in the

case of the latter this can extend to large portions of the face.

It would be reasonable to imagine that the cell wall would be a focus for attacking

this organism. In reality, whilst there have been studies of the biosynthesis of cell wall

materials, the precise molecular organization within the cell walls is largely unknown.

jS-glucans and chitin form a skeleton for the mannoproteins which are found both at the

outer surface and throughout the entire cell wall. By using wheat germ agglutinin it has

been shown that chitin is concentrated in the cross-walls between mother and daughter

cells, but is also distributed throughout the whole of the cell wall. It has not been

possible to examine the distribution of glucans using plant lectins because there is no

known lectin which reacts specifically with glucans. However, the use of a monoclonal

antibody that reacts with (l,6)-/5-glucan has enabled the linkages which connect (1,6)-

/3-glucan to mannoproteins and the distribution of (l,6)-/?-glucan in the cell walls to be

studied (Sanjuan et al. 1995). In S. cerevisiae, the synthesis of (l,6)-/3-glucan begins

early in the secretory pathway whereas in C. albicans it is apparently incorporated at a

later stage. In both yeasts, (l,6)-/3-glucan is located within an inner layer of the cell

wall which can be rendered accessible with tunicamycin.

In the yeast form, C. albicans could be confused with S. cerevisiae upon simple

microscopic inspection. However, major differences exist which would soon become

apparent even to one not familiar with yeast taxonomy. Candida albicans is diploid

and has no sexual cycle. This means that classical genetic methods like those described

for S. cerevisiae are not possible with this organism. Attempts to isolate mutants by the

use of mutagenic agents are almost bound to fail because of the improbability of

producing mutations in both copies of a given gene and nowhere else in the genome.

Naturally occurring mutants do, of course, exist. The more recent developments of

molecular genetics are now being applied to Candida, but the reader should not conclude

that this organism is understood to anything like the extent of S. cerevisiae. The isolation

of C. albicans genes homologous to those in S. cerevisiae by means of complementation

of S. cerevisiae mutants has been particularly useful as have techniques such as 'Ura

blasting' in which first one copy of a given Candida gene is disrupted with the coding

sequence of the URA3 gene and then the second copy is treated likewise. This process

can be applied sequentially to produce a strain with defined mutations in known genes

(Gow et al. 1993).

3.2 Alternative morphologies

One spectacular difference between S. cerevisiae and C. albicans is the ability of the

latter to switch to a hyphal pattern of proliferation (Gow 1994). A variety of different

factors and conditions have been described which can elicit this switch. These include

serum, neutral pH, certain temperature profiles, the addition of yV-acetylglucosamine

and many more (Odds 1988). In germ tube formation, a protuberance develops from

the cell and thereafter growth remains highly polarized (Fig. 2.7). The cell which

forms the tip of the developing germ tube remains polarized throughout the cell cycle.

It must be emphasized that this is hyphal growth where cell division is symmetric,

but in contrast to pseudohyphal development (where the next cell cycle is started

synchronously), in this case growth is asynchronous with the result that the apical cell

becomes progressively longer (see Fig. 2.5). It has not been shown that the virulence or

pathogenicity of C. albicans are uniquely due to either the yeast or hyphal form.

Nonetheless, the ability to be able to interconvert between the distinct morphologies

must surely be to its advantage. The hyphal form is considered to be specialized for

foraging (Kron & Gow 1995). Even if this is not the correct conclusion, it certainly

conveys the ability to penetrate tissue, whilst the yeast form would seem to be more

effective for dispersal, e.g. through the blood system. One of the justifications for

studying morphological switching in C. albicans is that this may reveal a unique target

for therapy or prophylaxis.

Another form of switching is well-known in C. albicans. This is the phenomenon

of 'phenotypic switching' (Soil 1992) whereby colony morphologies vary dramatically

(e.g. white, opaque, fuzzy, wrinkled.) These may seem trivial to the pharmacist or

physician, but the variability extends far beyond the mere appearance of the colonies.

It can encompass a vast array of biochemical alterations, antigenicities and drug

Yeasts and moulds 45

Fig. 2.7 Germ tube formation by

Candida albicans. For simplicity

the diagram merely illustrates the

nuclear content of the parental

yeast cell and the developing

germ tube. In real life there is a

complex rearrangement of

cytoplasmic constituents which

results in all parts except the

apex becoming highly

vacuolated.

sensitivities. Furthermore, it is documented that patients who have suffered from repeated

vaginal candidosis have yielded the same strain which has presented an alternative

phenotype on each occasion (Soil et al. 1989). Thus, this phenomenon seems to represent

a mechanism which has evolved to enable C. albicans to escape destruction by the

immune system. Phenotypic switching is so-called because it has been assumed that a

mutational event could not be responsible due to the high frequencies (up to 10%)

Fig. 2.8 Mating type switching in Saccharomyces cerevisiae. The founder cell (F) is a virgin (i.e. has

not formed a bud previously), carries the HO gene and is mating type a. After the first cell cycle there

will be the mother cell (M) and the daughter (D), both of which are mating type a. Mother cells which

carry HO are able to switch mating type whilst in the Gl phase of the cell cycle. Assuming that M

switches to mating type a, the progeny which result from M (a mother and a daughter) will thus both

be mating type a. Daughters cannot switch mating type, hence D will produce two cells of mating

type a. Note that two of the cells present at the four cell stage are mothers and hence capable of

switching mating type before entering the next budding cycle.

46 Chapter 2

observed. However, there clearly has to be a genetic basis to such variation. In S.

cerevisiae, mating type switching can occur (Herskowitz et al 1992) due to the presence

of the HO gene. This gene confers on a haploid strain of either mating type the ability

to switch to the opposite mating type. The opportunity to switch is only available to

mother cells which are in the Gl phase of the cell cycle. Thus, it is quite easy to calculate

that a single cell that was (say) mating type a could give rise after two complete cell

cycles to a colony that comprised two cells of mating type a and two of mating type a

(Fig. 2.8.) Hence, one could say that mating type switching in S. cerevisiae has a

frequency of approximately 50% in appropriate strains, i.e. even higher than the

frequency of phenotypic switching in C. albicans. The molecular basis of phenopic

switching remains unclear at the time of writing.

4 Cryptococcus neoformans

Cryptococcus neoformans is an encapsulated yeast which causes cryptococcosis, a

subacute or chronic infection of the central nervous system. In extreme cases, tissue

damage can also occur in the skin, bones and internal organs. Cryptococcal meningitis

is very frequent in AIDS patients. Despite this, it is not a newly discovered organism

and its pathogenic capabilities have been known for many years. For example, in 1955

it was known that Cr. neoformans was the causative agent in 10% of all fatal human

mycoses in the USA (Emmons 1955). It does not form a pseudomycelium, neither does

it develop hyphae. Surely this yeast provides adequate proof that neither is necessary

to be pathogenic to any warm-blooded animal! The yeast cells are almost spherical and

in conditions of high osmolarity they produce a much reduced capsule, such that the

overall size is less than 5fm\ in diameter. This renders them small enough to remain as

dust in the atmosphere and be inhaled. This is reckoned to be the route of all infections.

The yeast is inhaled and carried to the alveoli of the lungs. When in the warm, moist

alveoli, the yeast cells regenerate their thick capsules with the consequent release of

polysaccharides and glycoproteins into the host's bloodstream, both of which serve as

virulence factors (Murphy 1996). The capsule products cause neutrophils to lose their

surface L-selectin, which is required for leukocytes to attach to endothelial cells before

moving from the blood system to the tissues. Since the leukocytes are not sent to the

site of infection in the tissue, the yeast escapes. The immune system is further confused

by yeast cell products in the bloodstream due to the induction of suppressor T

lymphocytes which attenuate immune responses. It is believed that the release of melanin

from the yeast also helps it to evade the immune system. Melanin is thought to act as an

antioxidant which thereby protects cryptococci from oxidative killing. With such an

armoury of virulence, the reader will appreciate why rapid identification of this organism

is so important.

5 Neurospora crassa

Neurospora crassa is a filamentous pink mould. It became famous to scientists due to

the work of Beadle and Tatum in the 1940s when they developed the 'one gene—one

enzyme' hypothesis. Its life cycle is shown in Fig. 2.9. Unfortunately, the full force of

fungal nomenclature comes into play when considering this organism. Aerial hyphae

Yeasts and moulds 47

Fig. 2.9 The life cycle of Neurospora crassa. The figure illustrates both the asexual cycle via

macroconidia and the sexual cycle. In the case of the latter, the diagram represents the

interaction between a male of mating type A and a female of mating type a. The trichogyne

grows towards the male. There is fusion, one male nucleus enters and pairs with a female nucleus.

Rounds of synchronous nuclear divisions result in dikaryotic (i.e. containing two nuclei)

cells. Karyogamy produces a true diploid which immediately undergoes meiosis and

ascosporogenesis. The cycle is completed by germination of the individual ascospores to found fresh

mycelia.

from the heterokaryotic (i.e. containing many separate nuclei) vegetative mycelium

produce either macroconidia or microconidia. The macroconidia contain several nuclei

and re-establish vegetative mycelium when they germinate. Each microconidium is

uninucleate; their role in the life cycle is to fuse with the trichogyne (a specialized

hypha of opposite mating type.) The trichogyne is carried on the protoperithecium,

which, as its name suggests, is the precursor to the perithecium (fruiting body). Inside

the perithecium, nuclear fusion takes place, followed by meiosis and further differen-

tiation to produce an ascus containing eight ascospores. When the ascospores germinate,

they produce haploid mycelia which can form heterokaryons by means of hyphal fusions

with mycelia of the opposite mating type.

It is instructive to consider the similarities and differences in the life cycle of

S. cerevisiae and N. crassa. Disregarding the obvious difference that the former is a

yeast whilst the latter is a mould, it should be noted that both fungi have a vegetative

haplophase. The diplophase of S. cerevisiae can proliferate, whereas in N. crassa the

diploid rapidly undergoes meiosis and ascospore formation. Both organisms can exist

in one of two mating types, each of which is controlled by a single genetic locus.

Neurospora crassa has truly male and female thalli (singular, thallus, is the vegetative

body of a fungus) which are morphologically distinct. Neurospora crassa is an obligate

aerobe, hence, elimination of oxygen will prevent its growth.

6 Penicillium and Aspergillus

The scientifically informed layperson is aware that Penicillium species are associated

with a number of beneficial products. These include the important antibiotic penicillin

(originally from P. notatum, but which soon came to be prepared from P. chrysogenum

because this species produces more: see Chapters 5 and 7), and the ripening of Stilton,

Roquefort and Camembert cheeses with strains of P. roquefortii (blue vein cheeses)

and P. camembertii (surface-ripened cheeses). However, many more would be surprised

to learn that today's commercial penicillin-producing strains all derive from an organism

which was originally isolated from a rotting Canteloupe melon. This fact emphasizes

the real ecological place of such moulds where, of course, decomposition of fruits and

vegetables is an essential part of the recycling of materials in the biosphere. Life could

not continue on our planet in the absence of decomposer organisms.

A less desirable characteristic of many moulds is the production of mycotoxins.

One group of mycotoxins, the aflatoxins, which are derived from decaketides, are a

particular cause for concern. Aflatoxin Bj is carcinogenic in animals and mammalian

cell lines in tissue culture. It has been linked to specific mutations in the human

tumour suppressor gene p53 thus causing primary hepatocellular carcinoma. Hence,

contamination by aflatoxins of food, feed, and medical, veterinary, pharmaceutical and

laboratory preparations has serious health and economic consequences. Aspergillus

parasiticus and A. flavus are notorious as producers of aflatoxins. As with other species

of Aspergillus, they are very widespread in the environment and, aided by their rapid

growth on a variety of substrates, they are commonly found as contaminants of all

sorts of materials. Members of the genus can give rise to a group of diseases known

collectively as aspergilloses. These includes allergies, toxicoses, tissue invasion and

local colonization. Aspergillus fumigatus is the most common cause of aspergillosis.

Not all members of the genus are wholly bad. Aspergillus oryzae has been used for

centuries in the production of soy sauce. Although this is of little consequence in the

West, the underlying technology became the foundation of the Japanese amino acid

and nucleotide business which have worldwide economic importance.

It appears that the various strains of Penicillium used in cheese production do not

produce any such toxins. Presumably this is an example of selection acting on the early

human cheese-makers: the folk who produced cheese which contained mycotoxins ate

their own cheese and died. Hence, they did not hand on their skills and strains of mould

to subsequent generations! Microbiological contamination by wild moulds always carries

the possibility of chemical contamination with their toxins, so we should not think of

Penicillium species as being of universal benefit to humankind. Indeed, direct infections

by Penicillium species have been reported to various parts of the human body including

the cornea, ear, respiratory tract, urinary tract and heart (following the surgical insertion

of artificial valves: Kwon-Chung & Bennett 1992).

Penicillium has no sexual cycle. The organisms merely produce conidia (asexually

produced spores) which are readily dispersed by slight draughts (Fig. 2.10). New growth

can commence after landing on a suitable substrate. The brush-like appearance is typical

Yeasts and moulds 49

Fig. 2.10 A typical Penicillium.

(Fig. 2.10). The organism Penicillium marneffei is said to be thermally dimorphic: at

25-30°C it produces colonies like any other of the genus. At 35-37°C it is yeast-like

(Larone 1995). It is endemic in South-East Asia and is reported as causing deep-seated

infections in both immunocompromised and normal individuals who have visited those

parts. The wider significance of this to fungal biology and especially to the taxonomy

of Penicillium species remains to be established, but the importance to human health is

already clear.

Epidermophy ton, Microsporum and Trichophyton

All three of these are dermatophytes, i.e. filamentous fungi which can utilize keratin

for their nutrition. Keratin is the chief protein in skin, hair and nail. Hence, all of these

organisms are responsible for superficial mycoses in mammals. It is often stated that

dermatophytes are the only fungi to have evolved which rely upon infection for their

own survival. This mistaken belief results from a view which is too human-centred and

neglects, for example, the presence of symbiotic fungi in the stomachs of ruminants.

The genus Microsporum contains several interesting species including M. audouinii,

which, in years gone by, caused epidemics of 'ringworm' in children, but rarely in

adults: M.ferrugineum appears to fulfil this role today; M. canis which infects children,

cats and dogs, but not adults—it is said that the children acquire the infection from

the animals; M. gypseum affects mainly lower animals; M. gallinae infects poultry and

humans; M. nanum can be common in pigs, but is rare in humans. The appearance of a

tinea ('ringworm') is the host's reaction to the proteolytic (protein degrading) enzymes

secreted by the fungus. In highly sensitized or hyper-allergic individuals this can be

very pronounced.

Epidermophyton floccosum infects the skin and nails but not the hair, whereas

different species of the genus Trichophyton display both geographical and anatomical

variations. For example, T. rubrum is currently the most common dermatophyte of

humans: it infects skin and nails but almost never hairy parts of the body; T.

mentagrophytes, which is frequently the cause of athlete's foot, can infect all parts of

the human body; the aptly-named T. tonsurans is the major causative agent of scalp

ringworm in the USA whereas T. megninii is hardly ever found in the Western world.

These varied distributions presumably reflect a complex matrix of variables including

climate, nutrition, age, physiological status, the proximity of animals and other aspects

of human lifestyle.

Certain identification of each individual dermatophyte requires great skill especially

in the case of Microsporum where there is considerable morphological similarity between

the species: hyphae are septate with numerous macroconidia which are thick-walled

and rough in most cases. Microconidia are usually present. Epidermophyton is broadly

similar except that microconidia are not formed. Distinguishing individual species

of Trichophyton from each other is less problematical, although an unwary observer

might confuse T. mentagrophytes with T. rubrum. Generally, in Trichophyton species,

macroconidia are rare, thin-walled and smooth; there are numerous microconidia.

Clearly, although these organisms only cause superficial infections, a rapid, genetic-

based identification system would be a boon.

8 References

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Viruses

1 Introduction

2 General properties of viruses

2.1 Size

2.2 Nucleic acid content

2.3 Metabolic capabilities

3 Structure of viruses

3.1 Helical symmetry

3.2 Icosahedral symmetry

4 The effect of chemical and physical

agents on viruses

5 Virus-host-cell interactions

6 Bacteriophages

6.1 The lytic growth cycle

6.2 Lysogeny

6.3 Epidemiological uses

7 Human viruses

7.1 Cultivation of human viruses

7.1.1 Cell culture

7.1.2 The chick embryo

7.1.3 Animal inoculation

8 Multiplication of human viruses

8.1 Attachment

8.2 Penetration and uncoating

8.3 Production of viral proteins and

replication of viral nucleic acid

8.4 Assembly and release of progeny

9 The problems of viral chemotherapy

9.1 Interferon

10 Tumour viruses

11 The human immunodeficiency virus

12 Prions

13 Further reading

Introduction

Following the demonstration by Koch and his colleagues that anthrax, tuberculosis

and diphtheria were caused by bacteria, it was thought that similar organisms

would, in time, be shown to be responsible for all infectious diseases. It gradually

became obvious, however, that for a number of important diseases no such bacterial

cause could be established. Infectious material from a case of rabies, for example,

could be passed through special filters which held back all particles of bacterial

size, and the resulting bacteria-free filtrate still proved to be capable of inducing

rabies when inoculated into a susceptible animal. The term virus had, up until

this time, been used quite indiscriminately to describe any agent capable of

producing disease, so these filter-passing agents were originally called filterable viruses.

With the passage of time the description 'filterable' has been dropped and the name

virus has come to refer specifically to what are now known to be a distinctive

group of microorganisms different in structure and method of replication from

all others.

General properties of viruses

All forms of life—animal, plant and even bacterial—are susceptible to infection by

Viruses 53