Impaired respiration and mucus production by P. aeruginosa as a
possible model for mucus production by those with cystic fibrosis[*]
by Ian MacL. Campbell (B.Sc., Ph.D.)
Preface
This investigation ended in 1989 and was never completed because of lack
of funding. At that time, out of frustration, I promised myself I would never
again think about cystic fibrosis (CF) or Pseudomonas
aeruginosa (P. aeruginosa). It has taken me more than twenty years
to lose that mind-set. In November of 2010 it was reported that a young woman
with CF was about to have a double-lung transplant. It was the same person who,
as a young child, had been selected (in the late 1980’s) as the poster child by
the then Canadian Cystic Fibrosis Foundation. Her image was used as part of the
campaign to advertise how the discovery of the CF gene would, within a few
years, lead to the cure. The report of
this woman’s transplant bothered me so much that I was compelled to review the
results of our incomplete study, thinking that we must be able to do better. In
what follows my goal is to present information, which I have never seen
reported, that may help someone learn how to protect her new lungs from P.
aeruginosa. Having recently developed a terminal affliction, old age, I
wish to distribute this information, with some suggestions, without further
delay.
Introduction
The initial bacterial invader of the
pulmonary tract of those with CF is usually Staphylococcus aureus. Prolonged infection by this organism
causes air-way obstruction. Eventually this organism is replaced by P.
aeruginosa which invades as a non-mucoid, transforms to the mucoid state
and frequently causes death. Isolates of the mucoid form are unstable and revert
to the non-mucoid state when cultured in vitro. For a study and
discussion on this instability see J. Govan (J. Med. Microbiol. 8 [1975]: 513-522). Our initial plan was to
compare the possible effect of various fatty acids on mucus production.
However, what was observed during our first experiment resulted in a change of
plans.
Materials and Methods
Two strains of P. aeruginosa were
studied: PAO-381 (non-mucoid) and PAO-579 (mucoid). These isolates were
obtained from the Dept. of Bacteriology, Faculty of Medicine, of Toronto. I was
told that they had been isolated by Prof. J. Govan, University of Edinburgh. For
these experiments, the nutrient broth was 15 gr. of powdered Tryptic Soy medium
(Difco U.S.A.) per 100 ml. of distilled water.
The regimen was as follows: At 2 PM, two 500 ml flasks containing 100 ml
of sterile broth were inoculated from the stock plates of both strains and then
incubated at 37 C in a shaking water-bath. This provided broth inoculum for the
experimental cultures.
For the experiments, 250 ml of nutrient broth was put in 1 L flasks.
After sterilization, the flasks were closed tightly with rubber stoppers. The
rubber stoppers were fitted with a septum (to allow inoculation, gas purging and
sampling) and passages for an oxygen- and a pH-probe. The flasks were sealed to
allow assessment of oxygen consumption and the possible effect of decreasing
oxygen supply on the cell cultures. At 10 PM, each flask was injected with 10
ml of the appropriate broth inoculum, purged to equilibrium with air and the
probes positioned in the broth. They were then incubated in the shaking
water-bath at 37 C. The oxygen content and the pH of the cultures were
continuously recorded. The flasks containing the remaining inoculum were left
on the bench over-night to be autoclaved in the morning.
Results and Discussion
The initial run was to check our procedure and familiarize ourselves
with the organism. By 8 AM the oxygen pressure in the sealed cultures had
dropped by about one-half and the broths were opaque. There was no change in pH
or a visible sign of mucus in these cultures. However, the appearance of the two
inoculum cultures that had been left undisturbed over-night had changed
drastically. What was observed caused a complete change in research plans. The
broths were now completely clear and were covered with a layer of dense highly
viscous orange-yellow mucus. Microscopic examination of the mucus and clear
broth showed that all of the cells were contained in the mucus. When these
flasks were swirled, which re-oxygenated the cultures, the mucus separated into
a few large clumps and turned green assuming the usual appearance of mucoid
cultures of this bacterium. Within 30 seconds of being placed back on the
bench, the mucus rose to the surface and the original yellowish color returned.
This treatment was repeated several times and always gave the same result. The
color change in response to the absence or presence of oxygen suggested that an
oxidation-reduction reaction was occurring in the mucus complex. (From previous
experience working with insect blood, I suspect that the yellow-green color
change occurred because the respiratory pigment biliverdin was present in the
mucus complex.) The cause of the oxidation was apparent but the cause of the
rapid reduction was not. Could there be an elevated supply of hydrogen-ion
stored in the system?
Meanwhile, the cultures that were being incubated in the shaking water
bath were monitored. Every hour samples of both cultures were prepared for both
light and electron microscopy. By 18 hours, when the oxygen saturation became
nearly stable at about one-third saturation, some of the bacteria had an
altered appearance. Instead of these cells having a sharply defined margin they
appeared fuzzy with strands of fibrous material extending outward. By 19 hours
many of these “fuzzy cells” had aggregated to form mucoid clumps and shortly
thereafter these clumps had enlarged enough to become visible as typical green
aggregates of mucoid P. aeruginosa. These aggregates also underwent the
green to yellow change in response to the presence or absence of oxygen.
Surprisingly, the pH of the cultures remained unchanged at about pH 7.0.
It then occurred to us that something in the cultures may be “storing”
hydrogen-ion (i.e. a strong buffer). To check this possibility, small samples
of mucus and mucus-free material, taken from each culture, were shaken into 10
ml of water (double-distilled and de-ionized) and changes in pH were recorded.
Whereas the pH of the non-mucus samples came into equilibrium at between 5 and
6, that of the mucus samples stabilized at between 1.5 and 3. Evidently there
was a sizable store of hydrogen-ion within the mucus complex. Several repeats of the experiment gave us
similar results. Because funds and equipment were limited, we decided to
restrict the remainder of the study to the mucoid strain PAO-579.
Since an Aminco dual wave-length spectrophotometer was available, the
oxidation-reduction response of the cytochromes to these conditions was
investigated. This spectrophotometer was designed specifically for the study of
opaque material such as mucus and bacterial suspensions. The wavelength
settings were at 410 nm., the isosbestic point, and 430 nm., the maximal
light-absorbing wavelength of reduced cytochrome-C. A 1.0 cm. cuvette was
three-quarters filled with a sample of mucus- or non-mucus containing culture diluted
(3:1) in 10% glucose solution. The sample was then aerated by vigorous shaking and
immediately placed in the spectrophotometer. Once the optical-density reading became
minimal, indicating that cytochrome oxidation was complete, the subsequent increase
in optical density as the cytochrome underwent reduction, as the dissolved oxygen
became depleted, was continuously recorded.
Fig. 1 is a diagrammatic presentation of the results of a study on the
mucus from a 24 hr. culture of the mucoid strain, PAO-579. Line 1 shows the
increase in optical density during reduction in a sample that had been aerated.
The maximum reading was interpreted as complete reduction of the cytochrome
(O.D. = 1). The procedure was then repeated on the same sample after the
addition of a few grains of succinate. In the Krebs cycle, surplus succinate
restricts the generation of hydrogen-ion by severely limiting the rate of its own
conversion to fumarate. Line 2 shows that with restricted hydrogen-ion
generation, a large additional portion of cytochrome was oxidized by aeration.
The addition of more succinate did not reveal more cytochrome. Moreover, the
addition of hydrogen-peroxide to a matched sample from the same culture gave
the same result, Line 3. This minimal reading was interpreted as complete
oxidation of the cytochrome (i.e. O.D.=0). The minimal density readings (Lines
2 and 3, compared to that for Line 1) indicate that approximately only 35% of
the total cytochrome was oxidized and 65% remained reduced with aeration alone.
When the same treatments were applied to samples of the same culture that
contained no mucus, only about 25% of the contained cytochrome remained reduced
when aerated. Apparently, the rate of reduction in the mucoid cells was much
greater than in those which had not yet become mucoid.
The
pattern of the reduction of the cytochrome, following its’ complete oxidation
in response to aeration following the addition succinate (Line 2) or hydrogen
peroxide (Line 3),was biphasic.
The cytochrome that had been revealed by these
additives underwent reduction first and, after a delay, that which was oxidized
by aeration alone underwent reduction. This indicates that the mucus contained
two different types of cells, one type generating hydrogen-ion at a considerably
higher rate than the other.
When these experiments were repeated with
cells from young (6 to 8 hr.) cultures, which contained no “fuzzy cells” or mucus,
it was found that with aeration still about 10% of the cytochrome remained
reduced. When samples of these cultures were aerated with either succinate or
hydrogen-peroxide present, so that the cytochrome was completely oxidized, the
pattern of the of the subsequent reduction was again biphasic indicating the
presence of the two different cell types. In this instance those with the lower
hydrogen-ion generation predominated. (NOTE: Increasing the quantity of
succinate did not reveal more cytochrome but prolonged the subsequent reduction
period. This was overcome by the addition of malate which allows the hydrogen-ion
generating cycle to bypass the succinate blockage. See Fig.1 Line 4.)
With
prolonged incubation at low oxygen pressure, the mucus appeared to reach
maximal density and viscosity at between 45 and 50 hrs. Within the “splash
region” of the flask walls, mucus strongly adhered and contained hard pellets of
material that required a steel spatula to remove them from the glass. Some of
these pellets were sectioned and observed under an electron microscope. The pellets
consisted of dehydrated mucus, surrounding masses of cells which were so
tightly packed that they resembled multi-cellular tissue, i.e. encapsulated P.
aeruginosa.
After between
85 and 90 hours of incubation approximately 90% of the cytochrome contained in
the free-floating mucus remained reduced upon aeration. We could not measure
the oxygen content because, as mucus adhered to the nylon guard surrounding the
membrane of the oxygen sensor, the reading dropped from about 25 to 0% saturation.
Evidently, the mucus severely restricts oxygen diffusion. The pH of the culture
remained at about 7.0. All of the foregoing experiments were repeated more than
10 times and gave very similar results.
In a few instances
the duration of the incubation period was extended beyond 50 hours. By about 65
hours, the quantity, viscosity and adhesiveness of the mucus appeared to
decrease. By
75 hours there was no visible mucus, only the pellets
on the wall of the flasks remained. When samples of these cultures were added
to de-ionized water the pH increased
to between 11 and12. This suggested that
the loss of mucoidy coincidental with high pH may result from de-animation of
protein. In older cultures carbohydrate and lipid could be depleted so that
protein became the sole source of carbon for the cells. The consequent
de-animation would result in the release of basic ammonia. Testing showed that the
addition of carbohydrate to older cultures did
restore the formation of mucus. Unfortunately, spectral measurements were not
taken on the cytochromes.
Knowing that, with impaired oxygen supply, surplus hydrogen-ion accrues
and mucus production is initiated, we planned to investigate treatments that
could reverse its production. However, it was now 1989 and funding was
exhausted. It is now 2011 and, prompted by the report of the lung-transplant of
the former CF poster child, I have turned back to my research and asked, how
could the excess production of hydrogen-ion relate to mucus production? After
studying various sources, especially Chapter 2 of Algal Physiology and Biochemistry (W.D.P. Stewart ed., Botanical
Monographs, Vol. 10, 1974, Univ. of California Press), my understanding of the
formation of mucus is as follows. The primary structure of the mucus of P. aeruginosa is alginic acid, a linear polymer
of acid sugar units (usually mannuronic but sometimes guluronic) connected through
an oxygen atom that links carbons 1 and 4 of the linear units. (Figure 2 is an
illustration of a section of polymanuronic alginic acid.) The synthesis of the
alginic acid is genetically regulated. However, the formation of mucus requires
the formation of weak hydrogen-bonds between adjacent polymeric chains. When
the carboxyl groups are not protonated they carry a strong negative charge and
accordingly are hydrophilic and repel each other so that hydrogen-bonding
cannot occur. However, protonation of a carboxyl group with a hydrogen-ion
neutralizes the charge and it becomes hydrophobic. As more of these charges along
the alginic acid chains are neutralized, more sugar-acid units will aggregate
in the hydrophobic domain. This allows inter-chain linkage by hydrogen-bonds
forming between a carboxyl group in one chain and a hydroxyl-group in an adjacent
chain. With manuronic units, the remaining hydroxyl-group, within a unit, hydrogen-bonds
with the ring oxygen in the next unit and thereby further increases the rigidity
and hydrophobicity of the chain. Thus, the more excess hydrogen-ion generated the
more carboxyl groups will be neutralized, the more hydrophobic the alginate will
become and the greater the opportunity for hydrogen-bonding between chains
which results in more and more viscous mucus being formed.
Could the
secretion of mucus by the bacterial cells function to excrete surplus hydrogen-ion
and thereby assist in regulating the pH within the cells? Perhaps a surplus of
intra-cellular hydrogen-cation indirectly activates translation of the gene for
alginic acid synthesis by displacing the di-cationic iron, which is known to
block its translation. This could be a feed-back mechanism for the excretion of
surplus intra-cellular hydrogen-ion that is generated in hypobaric-oxygen
environments and allows this obligate aerobe to survive and flourish under such
conditions. Moreover, the excreted mucus surrounds the cells and affords them
protection from hostile entities such phagocytes and antibiotics. This could
facilitate the invasion of lungs when ventilation is severely restricted by an existing
Staphylococcus aureus infection. Our
observation on the formation of pellets containing tightly-packed cells
suggests that with prolonged severe hypoxia the mucus completely dehydrates
encapsulating the bacteria. Encapsulation is known to occur in many species and
it provides complete isolation from hostile environments.
The accepted wisdom in the 1980’s was that when the supply of oxygen to
cells is decreased their cytochrome content decreases. Our research indicated
that, at least in P. aeruginosa, this decrease is apparent rather than
real. The addition of either succinate or hydrogen peroxide to the substrate
either to suppress the rate of reduction or increase the rate of oxidation revealed
all of the cytochrome by facilitating its’ complete oxidation. Evidently, what
does seem to change in response to low oxygen is increased capacity to generate
hydrogen-ion.
Mucoidy in P. aeruginosa is known to occur in three medical
conditions: cystic fibrosis, chronic bronchitis and 3rd degree burns.
A standard treatment for the latter is hyperbaric oxygen. We had planned an in
vitro study on the effect of hyperbaric oxygen in reversing mucoidy in this
bacterium. Treatments that assist in oxidizing the hydrogen-ions from the mucus
should increase hydration, decrease viscosity and thereby facilitate some clearance
of the air-ways. However, if our observations are correct, they will not
prevent the intracellular production of mucus. Only increasing the capacity of
the electron-transport system to utilize the surplus hydrogen-ion being
generated by the Krebs cycle will arrest mucus formation.
Our findings may relate directly to the pathology of cystic fibrosis.
With CF, as with P. aeruginosa, when the uptake and transport of oxygen
is impaired, the secretion of mucus is elevated and the mucus is unusually
viscous. Are the glycoproteins of human mucus similarly affected by an excess
of hydrogen-ion being generated because of restricted oxygen supply? It seems
to me that elevated hydrogen-ion in any organism could destabilize both cation
and anion regulation in a way that could modify normal biochemical and
physiological processes (e.g. elevation of sodium and chloride-ion in the sweat
and mucus blockage of the pancreatic ducts). A study of the oxidation-reduction
system in mitochondria of CF individuals might lead to a better understanding
of the disease.
Figure 1.
Reduction of cytochrome-C in mucoid cells following oxidation of
Pseudomonas aeruginosa (PAO-579).
Figure 2. Three oxygen-linked units of polymanuronic
alginic acid. (Adapted from W.D.P. Stewart, 1974. See text.)