Hugo W.B., Russel A.D.(ed). Pharmaceutical Microbiology

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Hammond S.M., Lambert P.A. & Rycroft A.N. (1984) The Bacterial Cell Surface. London: Croom Helm.

Hinkle P.C. & McCarty R.E. (1976) How cells make ATP. SciAm, 238, 104-123.

Nikaido H. & Vaara T. (1986) Molecular basis of bacterial outer membrane permeability. Microbiol

Rev, 49, 1-32.

Russell A.D. & Chopra I. (1996) Understanding Antibacterial Action and Resistance, 2nd edn.

Chichester: Ellis Horwood.

The references below refer to the subject matter in 5.6.

Microscopy, DEFT

Pettipher G.J., Mansell R., McKinnon C.H. & Cousins, CM. (1980) Rapid membrane filtration—

epifluorescent technique for direct inumeration of bacteria in raw milk. Appl Environ Microbiol,

39,423-429.

Denyer S.P. & Ward K.H. (1983) A rapid method for the detection of bacterial contaminants in

intravenous fluids using membrane filtration and epifluorescent microscopy. J Parental Sci Technol,

37, 156-158.

Flow cytometry

Shapiro H.M. (1990) Flow cytometry in laboratory microbiology: new directions. Am Soc Microbiol

News, 56, 584-586.

Microcalorimetry

Beezer A.E. (1980) Biological Microcalorimetry. London: Academic Press.

Impedance

Silley P. & Forsythe S. (1996) Impedance microbiology—a rapid change for microbiologists. J Bacterial

80, 233-243.

Bioluminescence

Stanley P.E., McCarthy B.J. & Smither R. (eds) (1989) ATP Luminescence: Rapid Methods in

Microbiology. Society of Applied Bacteriology Technical Series No. 26. Oxford: Blackwell Scientific

Publications.

Stewart G.S.A.B., Loessner M.J. & Scherer S. (1996) The bacterial lux gene bioluminescent biosensor

revisited. Am Soc Microbiol News, 62, 297-301.

General reference

Stannard C.J., Petit S.B. & Skinner F.A. (1989) Rapid Microbiological Methods for Foods, Beverages

and Pharmaceuticals. Society of Applied Bacteriology Technical Series No. 25. Oxford: Blackwell

Scientific Publications.

Yeasts and moulds

1 Introduction 4 Cryptococcus neoformans

2 Saccharomyces cerevisiae 5 Neurospora crassa

2.1 The life cycle

2.2 Metabolism and physiology 6 Penicillium and Aspergillus

2.3 Cell wall

7 Epidermophyton, Microsporum and

3 Candida albicans Trichophyton

3.1 Pharmaceutical and clinical significance

3.2 Alternative morphologies 8 References

Introduction

Yeasts and moulds are members of the fungi. Yeasts are characterized as being essentially

unicellular, whereas moulds are composed of filaments which en masse frequently

appear fuzzy or powdery. The familar budding yeast Saccharomyces cerevisiae, also

known as Baker's or Brewer's yeast, is usually thought of as the typical yeast. The

green mould Penicillium digitatum, a frequent spoiler of fruits such as apples or oranges,

and the bread mould Neurospora crassa will also be well-known to many. These latter

two organisms are properly considered as typical moulds. As is usually the case, however,

life is not completely straightforward for there are a considerable number of so-called

'dimorphic fungi' which can alternate between yeast-like and filamentous forms. One

such organisms is Candida albicans. To make matters more complicated, it has been

rediscovered that the would-be typical yeast S. cerevisiae can also form filaments

under a variety of different conditions (Gimeno et al. 1992). All of these fungi have

pharmaceutical and medical significance. The precise nature of this significance is

different in each case. For example, S. cerevisiae is generally regarded as a totally safe

organism suitable for use in human food and drink; the reason for its importance is

because it is by far the best understood eukaryotic organism on the planet. In contrast,

Cryptococcus neoformans has a variety of ways by which it can evade defence

mechanisms of the immune system, but is relatively little studied. In between these

two extremes are many yeasts and moulds, which are omnipresent in the environment,

in or on our foods, or a part of the normal flora of humans, but all of which can

opportunistically contaminate pharmaceutical preparations or cause post-operative

disease. All fungi pose a threat to immunocompromised individuals. This knowledge

should be weighed against a background of a general lack of suitable antifungal agents

(see Chapter 5). The approach of this chapter will be to first describe S. cerevisiae in

considerable detail because so much is known about it. Then, other yeasts and moulds

will be considered in turn, pointing out (where appropriate) significant differences

from S. cerevisiae or from each other.

Yeasts and moulds 35

Saccharomyces cerevisiae

Saccharomyces cerevisiae has a predominant place in the realms of cell biology and

molecular biology where it has become accepted as the universal model eukaryote.

The main reason for this is its genetic tractability. Traditionally, for reasons associated

with its importance to the food and drink industry, a great deal was known about the

biochemistry and physiology of this yeast. Later, with the advent of yeast genetics, a

vast range of well-characterized mutants became available. In turn, because S. cerevisiae

can be transformed and is readily amenable to genetic manipulation, this permitted

the isolation and characterization of many yeast genes. Ultimately, in mid 1996

the nucleotide sequence of the entire genome of the organism was reported. This

achievement is still only a far-off dream for molecular biologists studying most other

eukaryotic organisms. Nevertheless, it is possible to identify genes from other organisms

by means of genetic complementation in S. cerevisiae. Explained briefly, only one

piece of DNA from another organism will be able to substitute for a mutation in a

known gene in S. cerevisiae—this is a segment of DNA which carries the homologous

gene (i.e. codes for the same function) in the other organism. The availability of well-

defined mutants in S. cerevisiae combined with the facility of genetic manipulation

and this yeast's short generation time, make this a very rapid way to identify heterologous

(i.e. belonging to another organism) genes. Many of the latest concepts in cell and

molecular biology (e.g. concerning control of the cell cycle) have been developed and

tested in this organism. Naturally then, since it is the prime model eukaryote, it is also

the best understood fungus.

The life cycle

The life cycle of S. cerevisiae is shown in Fig. 2.1. It can exist both as a haploid (one

copy of each chromosome per cell) or as a diploid (two copies of each chromosome per

cell). Haploids exists as one of two sexes referred to as mating type a and mating type

a. When two haploid cells come close together they cause each other to arrest in the

Gl phase of the cell cycle. Each subsequently produces a special protuberance enabling

growth towards the mating partner. These somewhat abnormal looking cells are termed

'schmoos'. A haploid will only mate with another haploid of the opposite mating type.

This is achieved by the expression of specific oligopeptide mating pheromones

(hormones with brings about behavioual change in cells of the opposite sex) and the

possession of surface receptors only for the opposite pheromone (hence, mating type a

strains produce only a-factor and have receptors for a-factor, whilst mating type a

strains produce only cu-factor and have receptors for a-factor). The resulting diploid,

like the haploids from which it arose, is capable of repeated rounds of vegetative

reproduction.

The vegetative cell cycle of S. cerevisiae has received extensive attention. There

are many justifications for this. Firstly, the cell cycle in this organism has many

convenient 'landmarks' (Hartwell 1974, 1978; Pringle 1978) which make it very easy

to identify the exact point in the cell cycle at which a cell happens to be. Examples of

these landmark events include bud emergence, the size of the bud, mitosis (nuclear

division takes place through the neck between the 'mother' cell and the bud), and cell

Fig. 2.1 The life cycle of Saccharomyces cerevisiae.

separation. Other markers of cell cycle progress are also apparent to the more experienced

observer (Fig. 2.2). The reader will notice from Fig. 2.2 that the 'daughter' which is

formed is smaller than the mother cell from which it arose. There is a size control

which operates over initiation of anew cell cycle (Pringle & Hartwell 1981), and since

the mother is larger than the minimum size necessary to pass this control, but the daughter

is not, the consequence is that the mother cell can immediately start a new cell cycle,

whereas the daughter must first grow for a period until it is large enough. Hence, mother

and daughter do not proceed through the next cell cycle synchronously. The significant

extent of morphological change throughout the cell cycle provides another reason for

studying this yeast as the construction of defined morphology.

A third justification for studying the cell cycle of this yeast is that it affords

a convenient system in which to study cell polarity. Together with asymmetric cell

division (inherent in the S. cerevisiae cell cycle with the unequal sized mothers and

daughters), the development of polarity is crucial in many aspects of development and

differentiation. Furthermore, as explained in more detail later in this chapter, the correct

Yeasts and moulds 37

Daughter

Cell

separation

Unbudded cell

Nuclear

division

Chitin

ring

Bud

emergence

Bud

enlargement

Fig. 2.2 'Landmark' events in

the cell cycle of Saccharomyces

cerevisiae. Gl, S, G2 and M are

the classical phases of the

eukaryotic cell cycle.

development of polarity is an essential aspect in the life of most fungi. The development

of polarity and the resulting asymmetric division can be considered as five constituent

processes (Lew & Reed 1995) shown schematically in Fig. 2.3. These are:

1 the F-actin cytoskeleton;

2 the polarity of growth achieved by the way new cell wall material is arranged;

3 the location of 10-nm neck filaments;

4 formation of the cell 'cap';

5 the distribution of DNA and microtubules.

The construction of a yeast cell requires isotropic growth. Bud emergence is signalled

by the accumulation of secretory vesicles, the rho protein Cdc42p and a cap of

membrane-localized actin patches. Once the cap is established, subsequent bud

emergence is accomplished entirely by polarized growth. Following bud emergence,

the rings of 10-nm filaments remain at the mother/bud neck, whereas the proteins in

the cap concentrate at the tip of the bud where secretion takes place. Later, there is a

critical switch back to isotropic growth which brings about swelling of the bud. Most

of the proteins of the cap appear to disperse simultaneously with the apical/isotropic

switch. At cytokinesis, secretion is redirected to the neck and the proteins of the cap

redistribute to this region. Mutants have been isolated which distinguish between the

separate components and processes. In turn, the genes which the mutations have

identified have all been characterized.

The pattern of budding in haploids differs from that in diploids (Friefelder 1960).

Haploids grown in rich medium bud in an axial pattern, i.e. each new bud site is placed

adjacent to the previous one. In the same rich nutrient conditions diploids exhibit bipolar

budding, in this case choosing new bud sites at either end of the cell (Fig. 2.4). Under

a variety of other conditions, all presumably involving some form of nutrient limitation,

diploids will form pseudohyphae and haploids will form invasive filaments. As alluded

to earlier in this chapter, this represents a 'rediscovery' in the case of S. cerevisiae

because it had been known for a long time and forms part of the basic taxonomy. Its

significance had been ignored. This situation arose because the ability to form these

structures had been crossed-out of the genetic background of many academic strains

Fig. 2.3 The development of polarity and asymmetric division in Saccharomyces cerevisiae. The

diagram is reproduced in a slightly simplified form from the work of Lew & Reed (1995) with the

permission of Current Opinion in Genetics and Development, (a) The F-actin cytoskeleton: strands =

actin cables; (•) cortical actin patches, (b) The polarity of growth is indicated by the direction of the

arrows; (arrows in many directions signifies isotropic growth), (c) 10-nm filaments which are

assembled to form a ring at the neck between mother and bud. (d) Construction of the 'cap' at the

pre-bud site. Notice that the proteins of the cap become dispersed at the apical/isotropic switch, first

over the whole surface of the bud, then more widely. Finally, secretion becomes refocussed at the neck

in time for cytokinesis, (e) The status and distribution of the nucleus and microtubules of the spindle.

Notice how the spindle pole body (•) plays an important part in orientation of the mitotic spindle.

Fig. 2.4 The budding pattern in

haploid and diploid

Saccharomyces cerevisiae. The

original cell which formed a bud

is the mother (M). The daughter

cell (D) is shown remaining

attached as might be the case in

i colonies growing on the surface

of agar.

around the world. Pseudohyphae are chains of regular-shaped, elongated cells in which

unipolar budding predominates. The analogous situation in colonies of haploids growing

on solid media is the formation of invasive filaments which are capable of penetrating

Yeasts and moulds 39

the agar. The generally accepted view is that starvation of nitrogen is the signal for the

switch from the yeast to a filamentous form (Kron et al. 1994), although it has also

been shown that pseudohyphal growth is strictly oxygen-dependent (Wright et al. 1993)

and that limitation of oxygen during continuous cultivation can result in the formation

of pseudohyphae (Kuriyama & Slaughter 1995). In 1996 Dickinson showed that,

dependent upon the concentration used, 'fusel' alcohols, i.e. n-amyl, isoamyl alcohol,

etc., caused the formation of hyphal-like extensions or pseudohyphae in a wide number

of different yeast species which were being cultured in rich liquid media where the

cells would normally proliferate as yeasts rather than in any other form (Dickinson

1996). It seems reasonable to conclude that since fusel alcohols are produced when the

yeasts are under various conditions of nutrient stress, the many situations which have

been reported to induce pseudohyphal formation are triggered by fusel alcohols. As we

have already noted, yeast-form proliferation is asymmetric and asynchronous; in

contrast, as others have already observed (Kron & Gow 1995), pseudohyphal growth

is symmetric and synchronous and, as will become apparent later in this chapter, hyphal

growth is symmetric and asynchronous (Fig. 2.5).

The diplophase and haplophase are equally stable. Hence, in the presence of adequate

nutrients, both are capable of repeated rounds of vegetative growth and mitosis.

However, in the presence of a poorly utilized carbon source such as acetate, and usually

in the absence of a nitrogen source, diploid strains switch to the alternative developmental

pathway of meiosis and spore formation. This process of sporulation gives rise to

structures termed 'asci'. Each single ascus contains four haploid ascospores (usually

referred to simply as 'spores'). Sporulation in diploid strains of S. cerevisiae has been

studied as a simple unicellular model of differentiation because it involves the co-

ordination of a complex sequence of genetic, biochemical and morphological events

(Fig. 2.6). The developmental switch occurs only in the Gl phase of the cell cycle, in

normal (a/a) diploids which are respiratorily complete. Hence, it requires the co-

ordination of signals about the environment, about the physiological and metabolic

Fig. 2.5 Cell cycles resulting in yeast-form cells, pseudohyphae and hyphae. In many respects the

cell cycle of pseudohyphal cells is similar to that of yeast-form cells, except that in pseudohyphae

G2 is prolonged, thus larger daughter cells are produced which are identical in size to the mother

cell. Hence, mother and daughter are both sufficiently large to start the next cell cycle and so bud

synchronously. In hyphae the apical cell becomes progressively longer. The diagram is reproduced

from the review of Kron & Gow (1995) with the permission of Current Opinion in Cell Biology.

40 Chapter 2

Fig. 2.6 The morphological events of sporulation in Saccharomyces cerevisiae. (a) starved cell: V,

vacuole; LG, lipid granule; ER, endoplasmic reticulum; CW, cell wall; M, mitochondrion; S, spindle

pole; SM, spindle microtubules; N, nucleus; NO, nucleolus, (b) Synaptonemal complex (SX) and

development of polycomplex body (PB) along with division of spindle pole body in (c). (d) First

meiotic division which is completed in (e). (f) Prepararation for meiosis II. (g) Enlargement of

prospore wall, culminating in enclosure of separate haploid nuclei (h). (i) Spore coat (SC) materials

produced and deposited, giving rise to the distinct outer spore coat (OSC) seen in the completed

spores of the mature ascus (j). Reproduced from the review by Dickinson (1988) with permission

from Blackwell Science Ltd.

status of the cell along with a way of monitoring the cell's ploidy and position in

the cell cycle. It is attractive for study because it involves meiosis, a relatively rare

event that occurs only in cells of specialized tissues in higher eukaryotes, and because

it allows the study of developmentally regulated gene expression. Transfer to the

sporulation pathway also involves a number of distinct metabolic switches (Dickinson

1988; Dickinson & Hewlins 1991). The whole process can be completed within 24

hours. The products of sporulation (haploid ascospores) have far greater resistance to

heat, solvents, dehydration, etc. than vegetative cells. If returned to good nutrient

conditions the spores will germinate and commence proliferation as free-living haploids.

This whole sequence of events forms the basis of conventional genetics and laboratory

strain construction in this yeast. Two haploids of opposite mating type each carrying

particular mutations are placed in close proximity to each other on the surface of an

agar medium. After mating and subsequent formation of the diploid, the cells can be

replica plated to a different medium to allow selection for the diploid and against the

parental haploids. ('Replica plating' involves making a replica of a group of cells which

Yeasts and moulds 41

are growing on one type of medium onto one, or more, different media. This is done by

pressing the agar surface of a Petri dish carrying cells onto a sheet of sterile velvet.

Subsequently, other, uninoculated, Petri dishes can receive doses of these cells by being

pressed onto the surface of the velvet). This is most simply arranged by ensuring that

each parental haploid has different auxotrophic requirements, hence the resultant diploid

will be able to grow on a minimal medium (due to complementation) whereas neither

of the parents can. After 1-3 days growth, the diploid will then be replica plated again

onto a sporulation medium. When the asci have formed, the individual spore progeny

can be separately grown as individual clones (a clone is a group of cells which are

genetically identical). This final step is accomplished by enzymatic digestion of the

ascus wall followed by micromanipulation of the individual spores (a process known

as 'dissection'). Due to the fact that meiotic recombination took place during sporulation,

the spores will have different combinations of mutations to those present in the original

parents. The precise combination of mutations in each spore can be determined by

analysing the phenotypes.

Metabolism and physiology

Saccharomyces cerevisiae is normally described as a faculative anaerobe which means

that it is able to proliferate under either anaerobic or aerobic conditions. It is able to

utilize a wide range of mono-, di- and oligosaccharides, ethanol, acetate, glycerol,

pyruvate and lactate. The favourite carbon source is glucose and the preferred mode of

metabolism is fermentative using the Embden-Meyerhof pathway (EMP) resulting in

the formation of ethanol. Many aspects of metabolism and physiology in this organism

(not merely carbon metabolism) are subject to catabolite repression which in most

cases means glucose repression. In the presence of glucose, synthesis of the enzymes

necessary for disaccharide (sucrose and maltose) or galactose utilization and for growth

on non-fermentable carbon sources (ethanol, acetate, glycerol, pyruvate and lactate) as

well as mitochondrial development are repressed. As the repressing substrate (glucose)

is consumed its concentration falls and the cells are said to become 'derepressed'; this

occurs typically at glucose concentrations below 0.2%. In other words, induction of

respiratory enzymes and components of the mitochondrial electron transport chain

occurs. This metabolic switch takes place late in the exponential phase of a batch

culture. As the cells pass through the deceleration phase and enter the stationary

phase they will be fully derepressed and will start to consume the ethanol that was

produced earlier. This requires the full participation of the tricarboxylic acid (TCA)

and glyoxylate cycles for the complete oxidation of ethanol to carbon dioxide and

water. Cells utilizing any of the non-fermentable carbon sources are also carrying out

gluconeogenesis. The glucose-6-phosphate produced as a result of this gluconeogenesis

is used both for the production of storage carbohydrate (trehalose) and for 'shuttling'

around the hexose monophosphate pathway (HMP) for synthesis of ribose which is

required for nucleotide (and hence ultimately nucleic acid) biosynthesis. The importance

of the glycolytic pathway to S. cerevisiae cannot be overstated. This is underlined by

the frequently quoted figure that the enzymes of glycolysis represent 30-65% (depending

upon physiological conditions) of soluble protein (Fraenkel 1982). The storage material

trehalose is produced in large quantities during sporulation (Dickinson et al. 1983). It

confers to the spore the ability to withstand dehydration. A wide range of organisms

in low water environments utilize trehalose for the same purpose including most

insects and the remarkable drought-resisting resurrection plant Selaginella lepidophylla

which can survive protracted desiccation until rains return (Leopold 1986). Trehalose

does this by preventing phase transitions within membranes (Crowe et al. 1984). The

compound is now added to a considerable number of laboratory products in order to

extend their shelf-life and its use in pharmaceutical and medical materials including

plasma, blood-based products, whole cells and tissues is under active investigation for

the same reason.

Notwithstanding the foregoing, an important constraint on this otherwise meta-

bolically flexible organism is the fact that proliferation under truly anaerobic conditions

(something that is very difficult to achieve in the laboratory) is not possible without the

provision of unsaturated fatty acid and sterol (Andreasen & Stier 1953, 1954). These

are required for the assembly of membranes. Naturally, mutants defective in fatty acid

or sterol biosynthesis have such requirements, but so do mutants with defects in

porphoryin biosynthesis due to the involvement of haematin in the synthesis of both

groups of compounds. Wild-type S. cerevisiae do not take up sterol under aerobic

conditions. It is possible to supply a limited range of alternative sterols instead of

the yeast's usual ergosterol. The ability of such an alternative sterol to support growth

of anaerobic S. cerevisiae is a way of assessing the structural specificity of sterol

requirement (Henry 1982). Some yeast sterol mutants were isolated as auxotrophs

requiring ergosterol whilst others were obtained on the basis of resistance to the polyene

antibiotic nystatin. Polyene antibiotics alter membrane permeability by interaction

with specific membrane sterols (Cass et al. 1970; Norman et al. 1972; see Chapter 8)

and seem not to inhibit lipid synthesis. Hence, mutants resistant to polyene antibiotics

have been useful in identifying the effects of altered sterol composition on different

membranes within the cell. This can be reflected in, for example, altered permeability

to a specific molecule or ion.

2.3 Cell wall

The cell wall of S. cerevisiae, like that of other fungi, is very strong. Despite its great

strength, one should remember that the cell wall is a dynamic structure (unlike a brick

wall). There are three major components:

1 an internal glucan layer,

2 the external layer of mannoproteins;

3 chitin which occupies various specialized locations.

Cell wall composition varies according to physiological conditions and developmental

status. For example, the wall of cells from stationary phase is much more resistant to

degradation by /3-glucanase than that from exponential phase cells (Necas 1971). The

glucan of S. cerevisiae is mainly j8(l-3)-linked glucoses with branching via /?(l-6)-

linked glucose units (Manners et al. 1973a, b). Most of the mannoproteins can only be

released after enzymatic degradation of the glucan layer. There are long «(l-6)-

mannose chains with a{\-2) and cu(l-3) side chains iV-linked to asparagine. There are

also short mannose chains O-linked to serine or threonine (Van Rinsum et al. 1991).

The carbohydrate chains of the mannoprotein layer are the main antigenic determinants

Yeasts and moulds 43