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LF223: Microbial Pathogens > Lecture 2. pH Tolerance > Flashcards

Lecture 2. pH Tolerance Flashcards

1
Q

What is the natural variance of pH within the environment?

A

Most acidic soil pH: ~0.5
Most alkaline lakes pH ~10.5

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2
Q

What does a pH of 0 mean?

A

1 mole per litre H⁺, 10⁻¹⁴ moles per litre OH⁻

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3
Q

What does a pH of 14 mean?

A

10⁻¹⁴ moles per litre H⁺, 1 mole per litre OH⁻

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4
Q

What is the usual range of pH units that most free-living prokaryotes grow over?

A

3 pH units

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5
Q

What is the main difference between growth rate vs temperature graphs and growth rate vs pH graphs?

A

The graphs are symmetrical above and below the optimum pH for growth

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6
Q

What are acidophiles?

A

Microorganisms which grow at an optimum pH well below neutrality (7.0)

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7
Q

What are neutrophiles?

A

Microorganisms which grow best at neutral pH

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8
Q

What are alkaliphiles?

A

Microorganisms which grow best under alkaline conditions

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9
Q

What are obligate acidophiles?

A

Microorganisms that can only grow at a low pH since their membrane membranes dissolve and the cells lyse at neutrality (thus a high [H⁺] is required for membrane stability)

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10
Q

What are examples of obligate acidophiles?

A

Some Thiobacillus species
Several genera of Archaea, including Sulfolobus and Thermoplasma
The eukaryotic alga Cyanidium (can grow at a pH of 0)

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11
Q

What are obligate alkaliphiles?

A

Can grow around pH 10 e.g in soda lakes & high carbonate soils

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12
Q

How do obligate alkaliphiles grow?

A

In an alkaliphilic Bacillus species a Na⁺ gradient (instead of the pmf) supplies the energy for transport & motility but a pmf can be established & drives respiratory ATP synthesis (even though the external surface of the membrane is so alkaline)

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13
Q

For most organisms, why does the intracellular pH need to stay around neutrality?

A

To prevent destruction of acid or alkali labile macromolecules

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14
Q

What are the documented extremes of internal pH within an acidophile and an alkaliphile?

A

Acidophile: pH 4.6
Alkaliphile: pH 9.5

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15
Q

What is the point of incorporating buffers into a bacterial growth culture?

A

Maintains the optimum pH of the medium in the changing milieu of bacterial waste products that accumulate during growth

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16
Q

What do many pathogenic bacteria exhibit in terms of pH growth?

A

Many pathogenic bacteria exhibit a relatively narrow range of pH over which they will grow
Most diagnostic media for the growth and identification of human pathogens have a pH near 7

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17
Q

What are the three major aspects of cell function may be affected by acidic conditions?

A

The capacity for nutrient acquisition and energy generation
Cytoplasmic pH homeostasis
Protection of proteins and DNA

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18
Q

Why is protection of proteins and DNA critical and what are two mechanisms for achieving this protection?

A

Critical for cell survival
chaperone proteins and alkalisation of the periplasm are two mechanisms for achieving this. The latter mechanism is used by the gastric pathogen Helicobacter pylori

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19
Q

What is the periplasm?

A

The gap between the inner and the outer membrane

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20
Q

What are the characteristics of Helicobacter pylori?

A

Gram-negative curved rod
Highly motile with 4-7 polar flagella
Uses glucose, amino acids, organic acids as a C source
Internal pH 7.0-7.3
Small, 1.7 Mbp genome (1 of the first to be sequenced)

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21
Q

What diseases are caused by H. pylori?

A

Gastric and duodenal ulcers
Ulcers associated with development of gastric cancer

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22
Q

What is the reservoir of H. pylori?

A

The human stomach

23
Q

What are main virulence factors of H. pylori?

A

Flagella
Urease – helps neutralise the acidic pH of the stomach
Adhesins
Vacuolating toxin

24
Q

How can H. pylori be treated?

A

Antibiotics cure disease & prevent ulcer recurrence

25
Q

What were Koch’s postulates about H. pylori causing ulcers?

A

The organism is always present in animals suffering from the disease but not in healthy individuals
The organism must be cultivated in pure culture away from the animal
Such a culture when inoculated into susceptible animals should initiate the disease characteristics
The organism should be re-isolated from these experimental animals & cultured (after which it should be the same as the original organism)

26
Q

Why did Barry Marshall drink a culture of H. pylori?

A

To show that H. pylori caused an inflamed stomach as the bacteria was found around the lesion
There was no animal model so he became the animal model

27
Q

Because H. pylori is quite sensitive to low pH, what part of the stomach does H. pylori colonise?

A

The mucin layer that covers the gastric mucosa (not the lumen of the stomach)

28
Q

How does mucus resist stomach acid?

A

Mucus resists diffusion of protons from stomach acid because it is composed of negatively charged sulphated polysaccharides (= mucus acts as a buffer to maintain a slightly alkaline pH at the mucosal surface)

29
Q

Why is motility such an important virulence factor for H. pylori?

A

Since H. pylori needs to reach the mucin layer to survive

30
Q

Because it takes time to reach the mucin layer, what do H. pylori require to survive?

A

Requires short-term pH protection via the enzyme urease

31
Q

What does urease do?

A

Urease hydrolyses the urea secreted by gastric cells to produce ammonia and CO₂. The ammonia neutralises stomach acid in its immediate vicinity
The urease is intracellular, so the organism takes up urea & exports ammonia very efficiently to the periplasm where the pH can rise to approx. pH 6

32
Q

In the gastric epithelium, what is the role of BabA?

A

Adhesin recognising Lewis b antigen which binds sulphated mucin sugars on epithelial cells

33
Q

In the gastric epithelium, what is the role of NAP?

A

Neutrophil activation protein – activates neutrophils
Leads to inflammation

34
Q

In the gastric epithelium, what is the role of VacA?

A

Vacuolating cytotoxin
Produces large vacuoles in mammalian cells

35
Q

What is UreI?

A

An inner membrane protein that facilitates urea entry in a pH-controlled way i.e. only when the incubation pH is acid
UreI consists of six transmembrane regions

36
Q

Where is UreI located and why?

A

The activity of UreI is controlled by external pH, so UreI is located on the periplasmic face of the membrane

37
Q

How do the periplasmic loops act as pH sensors?

A

The periplasmic loops and carboxy terminus contain a number of histidine and acidic residues that act as pH sensors through their ionisation

38
Q

How is internal pH managed by the diffusion of urea and H⁺?

A

Urea and H⁺ diffuse into the periplasmic space probably through porins in the outer membrane. Periplasmic acidification results in a conformational change in UreI, permitting the entry of urea into the cytoplasm, where it is rapidly hydrolysed, producing CO₂ and ammonia. The ammonia diffuses into the periplasmic space, where it becomes protonated, rasing the pH to about 6.5, a level consistent with survival and growth.

39
Q

What are the characteristics of Salmonella typhimurium?

A

Gram-negative rod
Motile

40
Q

What disease does S. typhimurium cause?

A

Gastroenteritis (one of the most common causes of diarrhoea)

41
Q

What are the reservoirs of S. typhimurium?

A

Human carriers, livestock animals, reptiles

42
Q

What is the mode of transmission for S. typhimurium?

A

Contaminated food – usually poultry meat or eggs (other documented sources: milk, ice cream & marijuana)

43
Q

What are the main virulence factors of S. typhimurium?

A

Adhesins
Invasion of mucosal cells (of small intestine)
Type III secretion system

44
Q

How is S. typhimurium prevented and treated?

A

Proper food handling; antibiotics

45
Q

How can S. typhimurium survive the acidic environment of the stomach?

A

If S. typhimurium cells are allowed to adapt (by growth at pH 6 for a generation) before being exposed to lower pH they can survive down to pH 3 (normally rapidly die off below a pH of 4)

46
Q

What allows S. typhimurium to survive the acidic environment of the stomach?

A

Fur (the regulator of iron acquisition genes) appears to regulate a subset of acid shock proteins. So, Fur senses pH as well as iron. Mutations have been identified in Fur that produce acid blind/iron-sensing & acid-sensing/iron blind phenotypes suggesting that iron and acid are sensed separately by this protein.

47
Q

Why is pH tolerance in Gram positive bacteria important?

A

Important in human health & economics e.g. survival and growth in food, effect on the oral cavity, gastric transit and intracellular survival

48
Q

What are three examples of pH tolerant Groups in Gram positive bacteria?

A

mutans Streptococci
Listeria
Rhodococcus equi

49
Q

How does mutans Streptococci achieve pH tolerance?

A

Gives rise to initiation and progression of dental caries; organisms produce acid and can grow at low pH

50
Q

How does Listeria achieve pH tolerance and surviving acid stress?

A

Requires a drop in pH to activate haemolysin, the toxin that permits its escape from the phagosome; acid adaptation means it can survive acidic foods such as cottage cheese, yoghurts, orange juice and salad dressing

51
Q

How does Rhodococcus equi achieve pH tolerance?

A

Particularly acid resistant and so can survive the low pH in alveolar macrophages

52
Q

What are the mechanisms of acid resistance in Gram positive bacteria?

A

Proton pumps
Protein repair/DNA repair
Regulators : e.g. several 2CSs in Listeria including LisRK
Altered metabolism: e.g. often of glycolytic enzymes, to facilitate rapid growth recovery when pH stress removed
Cell density: a quorum sensing system & biofilm growth allow for increased acid tolerance in S. mutans
Envelope alterations: e.g. S. mutans has increased levels of mono-unsaturated and longer chain fatty acids at pH 5 than 7
Production of alkali : e.g. urease production

53
Q

What are two examples of proton pumps involved in acid resistance in Gram positive bacteria?

A

F₁F₀-ATPases from tolerant bacteria are less sensitive to low pH
Glutamate decarboxylases (GAD) : consume protons via glutamate decarboxylation; the reaction product γ-aminobutyrate (GABA) is exported from the cell

LF223: Microbial Pathogens (15 decks)

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