Pseudomonas Article Part 2

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Results

P. aeruginosa is intraluminally located in CF airways. Analyses of CF

lungs revealed evidence for growth of P. aeruginosa predominantly as

spherical intraluminal colonies (Figure 1) (14). We have elected to refer

to these spherical colonies as " macrocolonies, " because many are visible

(>100 µm in diameter) to the naked eye. We used three separate techniques

to test whether macrocolonies were localized to intraluminal

mucus/mucopurulent material versus epithelial cell surfaces in CF lungs.

First, immunolocalization in thin sections from nine different CF lungs

revealed most bacteria (94.5%) were localized within the airway lumen

5–17 µm distant from the epithelial cell surface and the remainder in the

zone 2–5 µm from the epithelial surface (Figure 1c). None were identified

in contact with epithelial cells. Second, no P. aeruginosa bacteria were

observed attached to airway epithelia in TEMs of bronchi from nine CF

patients, containing a total length of 300 mm of bronchial surfaces.

Third, scanning electron microscopy detected no bacteria on bronchial

epithelia from two patients (total surface area examined: 116.1 mm2).

  Figure 1. P. aeruginosa is localized in intraluminal material of

freshly excised CF airways and binds to mucus. (a) Thin section of an

obstructed CF bronchus, stained with hematoxalin/eosin. Note the absence

of P. aeruginosa on epithelial surface (black arrow) and presence of P.

aeruginosa macrocolonies within intraluminal material (white arrows).

Blue gap is an artifact due to fixation. ( P. aeruginosa within

macrocolonies in a lung section, stained with rabbit Ab’s against P.

aeruginosa. Bars: a, 100 µm; b, 10 µm. © Percentage of bacteria

detected at a distance of 2-5 µm or 5-17 µm from the epithelial surface

of lungs from nine CF patients. Shrinkage artifacts were subtracted from

calculated distances. (d) Scanning electron micrograph of mucus-coated

spheroid derived from CF respiratory epithelium. P. aeruginosa (white

arrow) were enmeshed in mucus (black arrows) following a 2-hour

incubation. (e) Immunofluorescent staining of mucins (anti-mucin Ab)

bound to P. aeruginosa strain PAO1 in vitro. (f) Spheroid with adherent

mucus removed by prewash, then incubated with P. aeruginosa for 2 hours.

Note the absence of bacteria on ciliated epithelial cell surfaces. Bars:

d, 0.6 µm; e, 4 µm; f, 2.5 µm. Quantitative comparisons of PAO1 binding

revealed higher binding to mucus-coated NESfrom normal subjects (21.3 ±

10.6 bacteria/NES) versus non-mucus-coated (washed) NES (7.1 ± 0.1

bacteria/NES) (n = 6; 3 normal subjects; P < 0.05). Importantly, these

values were not different for CF NESs (26.4 ± 4.1 bacteria/NES for

mucus-coated NESs; 7.7 ± 3.9 bacteria/NES for washed NESs).

P. aeruginosa binding to mucus versus airway epithelial cell membranes in

vitro. We also tested the hypothesis that P. aeruginosa binds to mucus

rather than airway epithelial cells in vitro. Nasal epithelial spheroids

(NESs) spontaneously produce mucin, a fraction of which adheres to

ciliated cells (9). Incubation of P. aeruginosa PAO1 with NESs revealed

that the bacteria were enveloped by the mucus attached to NESs (Figure

1d). Mucin binding was also demonstrated by incubating P. aeruginosa with

mucins secreted by NESs (Figure 1e). In contrast, washing NESs to remove

adherent mucins greatly reduced P. aeruginosa binding (Figure 1f). O2 is

depleted within Pseudomonas-infected intraluminal mucopurulent masses in

vivo. The pO2 in CF mucopurulent masses in vivo was measured by inserting

an O2 electrode directly into the right upper lobar bronchi of

chronically infected CF patients (Figure 2a). When the probe was in the

bronchial lumen, the pO2 reached approximately 180 mmHg, a value

consistent with the supplemental O2 administered during bronchoscopy.

Upon probe insertion into the mucopurulent material obstructing the lobar

bronchus, the pO2 declined rapidly to a mean value of 2.5 mmHg (Figure

2b).

  Figure 2. Oxygen partial pressure (pO2) CF airways in vivo and in thick

films of ASL on human airway epithelial cultures. (a) pO2 in CF airways.

First 30 minutes represents measurement in a nonobstructed region of the

airway lumen. The arrow indicates insertion of oxygen probe into a

mucopurulent mass. The pO2 returned to basal values after probe

retraction from the adherent mass into the nonobstructed airway region.

( pO2 in nonobstructed CF airway lumens (L) and CF mucopurulent masses

(M) in vivo. n = 3 CF subjects; *P = 0.001. © Plots of pO2 gradients

under thick film conditions at 37°C in NL (squares; eight cultures/five

subjects) and CF cultures (circles; six cultures/four subjects). (d) pO2

gradients under thick film conditions measured at 4°C in NL (squares;

five cultures/three subjects) and CF cultures (circles; five

cultures/three subjects). (e) pO2 gradients in CF mucus that had

accumulated for 48 hours on CF culture surfaces and had become stationary

due to volume hyperabsorption (inverted triangles). Mucus transport was

restored in these cultures by addition of 30 µl PBS, and pO2 gradients

remeasured 1–2 hours later (triangles; six cultures; three subjects

each). (f) Comparison of pO2 gradients in NL (squares; nine cultures/six

subjects) and PCD (diamonds; five cultures/two subjects) cultures under

thick film conditions. Data are shown as mean ± SEM. *Significantly

different (P < 0.05) from pO2 at the air-liquid interface (0 µm).

Significant difference (P < 0.05) between NL and CF.

In vitro analyses of the genesis of O2 gradients in uninfected ASLs. The

diffusion of O2 through liquids is slow compared with air (15) so that

the hypoxia measured within the mucopurulent luminal masses could

reflect, in part, restricted O2 diffusion through thickened intraluminal

liquids. Although bacteria or neutrophils likely consume O2 and

contribute to the low pO2 measured in mucopurulent lumenal masses in vivo

(see below), it is possible that the O2 consumption of the underlying CF

epithelium uniquely contributes to mucus O2 gradients before infection.

To test the hypothesis that airway epithelial O2 consumption generates O2

gradients in liquid films that mimic in height mucus accumulated on CF

airway surfaces (16), we measured O2 gradients in NL and CF airway

epithelia covered by an approximately 800-µm thick ASL (PBS). NL airway

epithelia generated measurable O2 gradients at 37°C in this layer (Figure

2c). However, the pO2 gradient was significantly steeper in CF cultures

(Figure 2c). Both NL and CF ASL O2 gradients were abolished at 4°C,

suggesting that the gradients indeed reflected epithelial O2 consumption

(Figure 2d). Next, we asked whether the presence of mucins within ASL and

mucus transport were important determinants of O2 gradients under these

thick film conditions. No differences in O2 gradients were observed in CF

cultures with or without rotational mucus transport (Figure 2e). Thus, we

conclude that neither the presence of mucus, nor mixing, which occurs as

a consequence of mucus transport in situ (17), are important contributors

to O2 gradients measured within ASL. Rather, the gradient reflects ASL

depth and rate of epithelial O2 consumption. Do CF airway epithelia

generate disease-specific steeper O2 gradients within ASL? We measured

the O2 gradients in cultures derived from patients with primary ciliary

dyskinesia (PCD), a genetic disease of ciliary motility characterized by

chronic airways infection (18). The pO2 gradients in PCD ASL resembled

that of normal cultures and were shallower than CF ASL gradients (compare

Figure 2, f and c). Thus, it appears that the ability to generate steep

O2 gradients within ASL reflected a unique feature of CF airway

epithelia. Will bacteria deposited on mucus surfaces penetrate to hypoxic

zones in mucus layers? If bacteria contained within inhaled droplet

aerosols deposit and remain on the surface of the mucus layer, then

bacteria would be persistently exposed to normoxic environments.

Conversely, if inhaled bacteria move ( " swim " ) into the mucus layer, they

may face low pO2. Two sets of experiments were designed to distinguish

between these possibilities. First, we examined the behavior of bacteria

contained in small volumes (25 nl), designed to mimic aerosol droplets,

deposited on the surface of CF airway epithelial cultures that exhibited

rotational mucus transport. At the earliest time point measurable (3

minutes), motile P. aeruginosa penetrated into the mucus layer (Figure

3a). We next asked whether bacterial penetration into mucus reflected

bacterial motility or mucus mixing in the vertical axis during rotational

surface (horizontal) mucus transport (17). Fluorescently labeled beads

deposited on the airway surface exhibited similar kinetics of penetration

into the mucus layer, suggesting turbulent flow within the mucus layer

accounted for penetration to hypoxic zones (Figure 3b).

  Figure 3. Localization of P. aeruginosa and beads in transported and

stationary ASL (mucus) produced by planar CF cultures. Representative

confocal images of ASL (red) fluorescent P. aeruginosa (green) or green

fluorescent beads. P. aeruginosa or beads were added to the air-liquid

interface in 25-nl aliquots by a microsyringe mounted in a hydraulic

micromanipulator. (a) X-Z confocal image of P. aeruginosa 3 minutes after

addition to the surface of ASL (mucus) exhibiting rotational transport.

( X-Z confocal image of beads 3 minutes after addition to the surface

of mucus exhibiting rotational transport. Note that due to the rapid

" tumbling " movement of the mucus it was not possible to obtain early

time-point images of P. aeruginosa or beads at the air-liquid interface.

P. aeruginosa 3 minutes © and 15 minutes (d) after addition to

stationary mucus. Beads at 3 minutes (e) and 15 minutes later (f) after

addition to stationary mucus. Scale bars, 100 µm.

Second, we tested whether motile P. aeruginosa could penetrate mucus

masses adherent to CF airway surfaces. Within 15 minutes, P. aeruginosa

had penetrated deep into the mucus (compare Figure 3, c with d). In

contrast, fluorescent beads remained on the surface of the mucus plaque

(compare Figure 3, e with f), suggesting that bacterial motility was

required for P. aeruginosa penetration into hypoxic zones within

stationary mucus masses. Response of P. aeruginosa to a hypoxic

environment. P. aeruginosa is an aerobic bacterium that will grow under

anaerobic conditions if sufficient terminal electron acceptors are

provided (19-21). Because it is not yet known what bacterial culture

media best mimics human ASL (mucus), we tested for Pseudomonas growth

under aerobic versus anaerobic conditions, using ASL harvested from CF

and NL cultures. P. aeruginosa grew equally well in aerobic and anaerobic

conditions (Figure 4a).

  Figure 4. Growth and alginate production of P. aeruginosa under aerobic

versus anaerobic conditions. (a) Growth of P. aeruginosa in NL or CF ASL

under aerobic or anaerobic conditions. Two strains, PAO1 (black bars) and

ATCC 700829 (gray bars), were inoculated (100–200 bacteria, dashed line)

in 30 µl NL or CF ASL and number of bacteria quantitated 72 hours later.

The results presented are from a single representative experiment of

three performed. The differences in the CFU/ml in the three experiments

were less than 0.3 log 10. ( Immunofluorescence detection of alginate

production by PAO1 after aerobic (8 hours; left) or anaerobic (12 hours;

right) conditions (magnification, x1,000; bars, 10 µm). © Alginate

production of PAO1 by the carbazole assay after growth under anaerobic

(black bar) or aerobic (white bar) conditions for 4 days without added

nitrate. *P < 0.05. (d) Alginate production per microgram bacterial

protein mass of PAO1 as a function of the added NO3– to PIA under aerobic

(white bars) and anaerobic (black bars) conditions. (e) pO2 (filled

squares) in an aerobically growing suspension of P. aeruginosa (open

triangles) as a function of time (hours). (f) Mathematical analysis of

depths from air-mucus interface at which pO2 becomes zero for simulated

mucus masses/plaques containing different concentrations of P. aeruginosa

bacteria (colony-forming units per milliliter).

To test whether growth of Pseudomonas under anaerobic conditions in ASL

was supported by NO3– as a terminal electron acceptor, total nitrate

concentration was measured and found to be not different in uninfected

ASL from NL (20 µM) and CF (26 µM) cultures. These ASL total nitrate

concentration values are substantially lower than those reported for

airway secretions collected in vivo. For example, tracheal secretions

from control subjects have been reported to contain 144–421 µM total

nitrate (22, 23). The higher levels from tracheal secretions could

reflect the fact that upper airways produce more NO than the lower airway

regions (24, 25) that are representative in our cultures (third to sixth

generation bronchi). Importantly, as we found in ASL from NL and CF

cultures, no differences in tracheal secretion total nitrate

concentrations were found between " stable " CF patients (range 387–421 µM)

and controls (22, 23). We next asked whether the stress of anaerobic

environments could induce P. aeruginosa to acquire phenotypic features

that allows it to evade host defenses. Therefore, we measured P.

aeruginosa production of alginate, an exopolysaccharide involved in P.

aeruginosa biofilm formation, under anaerobic versus aerobic conditions

on agar plates (Columbia sheep blood agar, 37 µM total nitrate

concentration, and PIA, 63 µM total nitrate concentration) that contained

total nitrates in concentrations similar to that in ASL. Both

immunofluorescence detection of alginate associated with the bacterial

surface (Figure 4b; note thicker alginate coat in right panel) and

quantitative measurement of alginate/bacterial protein mass (Figure 4c)

demonstrated increased alginate production when PAO1 was grown under

anaerobic conditions. A thicker alginate coat (50%) was also observed for

PAO1 grown in ASL under anaerobic versus aerobic conditions. To test

whether the behavior of PAO1 mimicked that of environmental strains that

may infect CF mucus early in the course of the disease, alginate

production by 15 nonmucoid P. aeruginosa environmental strains,

genetically different by pulsed field gel electrophoretic analysis, was

compared during growth in aerobic versus anaerobic environments. These

strains routinely produced more alginate under anaerobic (0.191 ± 0.037

µg alginate per microgram of bacterial protein) versus aerobic (0.022 ±

0.004 µg alginate per microgram of bacterial protein) growth conditions.

We next tested whether alginate production could, in part, reflect

" stress " of an anaerobic environment with limiting concentrations of

[NO3–] as a terminal electron acceptor. As shown in Figure 4d, the ratio

of alginate to bacterial protein mass was highest at lower nitrate

concentrations, including the 63 µM nitrate value of PIA (no added

nitrate) that is most similar to values in ASL. Finally, we asked whether

the introduction of bacteria as O2-consuming elements into mucus

contributed to the magnitude of the O2 gradients observed in vivo (Figure

2). O2 tensions were reduced when P. aeruginosa growing in an open glass

tube reached densities of approximately 5 x 106 to 5 x 107 CFU/ml (Figure

4e). At approximately 3 x 107 CFU/ml, virtually all O2 was consumed.

Modeling of bacterial O2 consumption (Figure 4f) showed that anaerobic

conditions are generated at very shallow depths (3 µm) in infected mucus

masses when bacterial counts exceed 106 CFU/ml. Thus, the cell-specific

O2 gradients within uninfected mucus accumulating on the CF airway

epithelial surfaces will be exacerbated by the introduction of P.

aeruginosa into mucus.

    Discussion

Our studies initially focused on the pathogenesis of established CF

airways infection and, taking clues from these studies, explored whether

these variables could uniquely contribute to the early pathogenesis of P.

aeruginosa infection in CF airways. Morphometric analyses of freshly

excised lungs by three techniques demonstrated that P. aeruginosa grows

as macrocolonies in the airway intraluminal rather than the epithelial

surface compartment (Figure 1, a–c). These findings contradict recent

hypotheses emanating from in vitro model systems that focus on

high-salt/defensin inactivation (26) or luminal epithelial cell binding

(4), which predict bacterial infection of CF airway epithelial cells

themselves (5, 6). However, our data are consistent with those from

animal models that have demonstrated the adherence of P. aeruginosa to

respiratory mucus (27-29), and three previous qualitative studies of CF

postmortem lungs that identified P. aeruginosa in airway lumens rather

than on airway epithelial cells (30-32). Furthermore, they are also

consistent with our studies of NSEs that revealed P. aeruginosa

preferentially bound to mucus rather than epithelial cell surfaces

(Figure 1, d–f). A k

Becki

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