African Journal of Biotechnology
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African
Journal of Biotechnology Vol. 1 (1), pp. 10-16, November 2002 ISSN 1684-5315 © 2002 Academic Journals
Accepted 9 September 2002 |
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| Abstract | ||||||||||
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Swarming motility, a multicellular behaviour
characterized by periodic concentric growth on solid media has severally
been reported as a constraint in the clinical investigation of
mixed-culture infections involving Proteus and as a requirement for
virulence. While media are being formulated to restrain swarming in this
organism, the roles played by amino acids in the biogenesis of swarming
have not been fully clarified. The effects of 20 amino acids on swarming,
extracellular protease activity, cellular RNA level and total protein
concentration in 20 clinical Proteus strains from Lagos, Nigeria
were investigated. At 0.5mM concentration, glutamine, serine, and
methionine were the only amino acids found to enhance swarming motility on
M9 minimal medium (7.1–11.3 mm), cause significant increases in protease
activity (33.7–48.3 units/ml), total protein concentration (22.5–28.6
mg/ml) and cellular RNA concentration (192.8–264.7 mg/ml)
when compared to values obtained for other 17 amino acids (P < 0.05).
Furthermore, the amino acid induced swarming in these strains was found to
vary directly and significantly (P < 0.05) with protease activity and
cellular RNA concentration. The exclusion of methionine, serine and
glutamine when supplementing antiswarming media for clinical investigation
of Proteus - associated polymicrobial infections in Nigeria is
suggested.
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| Introduction | ||||||||||
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Many recurrent cases of bacteruria and urinary
tract infections involving Proteus have been reported (Lopatkin et
al., 2001; Pfau and Sacks, 1992). The re-occurrence of these diseases have
been attributed to polymicrobial etiology through cultures, which is
sometimes undetected (van Asten and Gaastra, 1999). Proteus exhibits
swarming motility, a growth behavior that
may overwhelm
the growth
of other
pathogens thereby annulling plans for an adequate antibiotic
regimen options. Proteus swarming behavior could be refrained on minimal
media supplemented with substances such as urea, activated charcoal,
sodium azide, nitrophenylglycerol
and alcohol (William, 1973; Hernandez and Cavallo,
1999). However, the effects of these
substances on other pathogens remain largely unknown. Although minimal
media are supportive to the growth of Proteus and Pseudomonas
species (Dick et al., 1985), it is grossly inadequate to meet the growth
requirements of fastidious pathogenic microorganisms (Marshall et al., 1984; Reynolds
and Penn, 1994). In this regard media fortification with
amino acids that will promote growth of pathogens but inhibit swarming is
highly desirable. Studies on swarming
enhancing effects of amino acids in Nigeria are scarce but available data
from studies conducted elsewhere, revealed variability in the ability of
polar and non-polar amino acids to support swarming motility in Proteus
species (Ulitzer, 1975; Eberl et al., 1999). Swarming motility has also
been linked to virulence in Proteus.
Allison et al. (1992a) showed the dependence of Proteus
mirabilis invasion of urothelial cells on motility and swarming
differentiation. In the present study, the swarming
modulatory effects
of 20 amino acids
were investigated. In order to provide biochemical evidence
for this behavior in relation to virulence, cellular RNA, extra-cellular
protease activity and level of total protein in 20 Proteus strains
were also determined.
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| Materials and Methods | ||||||||||
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Proteus strains Twenty clinical Proteus strains that
displayed swarming motility on nutrient and blood agar media were
recruited for study. They were recovered from agar slants in the Genetic
and Biotechnology Department of the Nigerian Institute of Medical
Research. Yaba – Lagos. Before use, the strains were subjected to
biochemical characterization for clarification (Cowan, 1974). Effects of amino acids on Swarming
motility 20ml of an overnight culture of Proteus
in LB broth was subcultured in a test tube containing 10ml of M9 minimal
media (Na2HPO4, 1.28%; KH2PO4,
0.31%; NH4Cl, 0.1%; Glucose, 0.4%) supplemented with 0.5 mM of
each of the 20 amino acids tested. They
were glutamine (Gln), glutamate (Glu), aspartate (Asp), asparagines (Asn),
tryptophan (Trp), tyrosine (Tyr), Phenylalanine (Phe), glycine (Gly),
cysteine (Cys), proline (Pro), leucine (Leu), valine (Val), methionine
(Met), lysine (Lys), threonine (Thr), alanine (Ala), isoleucine (Ileu),
histidine (His), arginine (Arg), serine (Ser). The inoculated tubes were
incubated aerobically at 37°C
for 24 hours. Meanwhile, at 8h incubation, 3 ml of culture was used to
inoculate another batch M9 minimal media but solidified with 0.6% Eiken
agar at the center. At 24 h, the outer diameter of swarming zone from the
point of inoculation was measured in millimeter. Broth cultures were also
centrifuged (3000 rpm, 10minutes, 0°C),
cell pellets were lysed and lysate was used for RNA estimation. Total
protein and protease activity were measured using the cell fee
supernatant. Total Cellular RNA Estimation The total cellular RNA was determined
following the reaction of cell lysate with orcinol reagent as described
by Jayaraman (1988). Initially,
the bacterial cells obtained as pellets of M9 minimal media culture were
dispersed in 200 ml of O.1 M Tris–EDTA buffer (pH 8.0) and then lysed by
adding 400 ml lysing solution (3% SDS in 50 mM Tris–HCl and 2 N NaOH)
for 5 min. Cell lysis was stopped by the addition of 300 ml sodium acetate
buffer (pH 5.5). The suspension was subsequently centrifuged (5000rpm) for
5 min at 4°C.
The supernatant obtained was used for total RNA estimation.
Cell-Free Protein Determination The method of Lowry et al. (1951) was
adopted to determine the concentration of total protein secreted by the Proteus
strains tested under the influence of amino acids. Bovine serum albumin
(500–1250 mg/ml) was employed as standard protein. Extracellular Protease Activity Protease activity was measured as described by Li and Yousten (1975) with some modifications. Overnight culture of Proteus in M9 minimal media was centrifuged (3000 rpm, 10 min, 0°C) and the resulting supernatant was filter–sterilized by passage through a 45 mM Millipore filter. 50 ml of the cell free filtrate was pipetted in a tube containing 1 ml of 0.5% bovine serum albumin (BSA) and 2 ml of phosphate–saline buffer (PBS, pH 7.2). After mixing, the assay mixture was incubated at 37°C for 1 hour. Digestion was terminated by adding 0.5 ml of 5% trichloroacetic acid (TCA). One unit of protease activity was defined as the amount of enzyme that caused a 0.01 increase in absorbance at 280 nm under the assay conditions.
Statistical analysis Data were expressed as range and mean + standard deviation. Differences between mean values were compared for significance using the student’s t – test. Relationships between parameters were assessed by regression analysis. P value < 0.05 was considered significant in all cases.
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| Results | ||||||||||
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Table 1 summarized the effects of the twenty amino acids at 0.5 mM on extracellular protease activity, swarming motility, total protein concentration and cellular RNA level in 20 Proteus strains grown in M9 minimal medium. Higher cellular RNA level was observed in media supplemented with glutamine (264.7 mg/ml), methionine (224.8 mg/ml) and serine (192.8 mg/ml) when compared with levels observed with respect to the remaining 17 amino acids (132–166.2 mg/ml). Similarly, higher concentration of total protein (22.5–28.6 mg/ml) and extracellular protease activity (33.7–48.3 units/ml) in glutamine, methionine and serine media compared with those supplemented with the remaining 17 amino acids (protease: 12.2–23.8 units/ml; protein: 9.4–14.2 mg/ml). Unlike in the glutamine, methionine, and serine supplemented media where outermost colony zone diameter (OCZD) range of 7.1–11.2 mm was observed, 13 of the remaining 17 amino acids resulted in OCZD £ 2 mm after 24 h of growth of the Proteus strains.
Table 1. Swarming motility and biochemical changes in Proteus strains grown in M9 minimal media supplemented with amino acids.
The observed increases of these biochemical parameters in the glutamine, methionine, and serine minimal media were further found to be significant (t = 8.44–12.02; P<0.05) (Table 2). Further comparison of these biochemical parameters between glutamine supplemented media and those fortified with methionine and serine indicated greater and significant (P < 0.05) enhancing effects of glutamine on protease activity (48.3 vs 33.7 and 38.2 units/ml), total protein concentration (28.6 vs. 22.5 and 26.1 mg/ml) and cellular RNA level (264.7 vs 192.8 and 224.8 mg/ml). Methionine supplementation subsequently produced significant elevation in these biochemical parameters compared with the effect of serine (t = 9.1–21.2; P < 0.05) (Table 3). Table 2.
Comparative evaluation of biochemical parameters in M9 minimal media
based on amino acid supplementation. 
Table 3. Variation in glutamine, methionine and serine induced increases in cellular RNA, total protein and protease activity among the Proteus strains.
Linear and significant
relationships were also observed between swarming motility and protease
activity (r =0.49, P < 0.05) and between swarming motility and cellular
RNA levels (r = 0.47; P < 0.05) among the Proteus strains grown
in serine, glutamine
and methionine
M9 minimal
media (Figure 1).
A.
B.
Figure 1. Swarming modulatory effects of serine, glutamine and methionine in Proteus. Proteus strains were grown in M9 minimal media supplemented with serine, glutamine and methionine. (A) Relationship between swarming motility and extracellular protease activity. (B) Relationship between swarming motility and cellular RNA level.
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| Discussion | ||||||||||
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The
survival and growth of the Proteus strains tested in M9 minimal
media further demonstrates the ability of this organism to biosynthesize
growth factors from simple nitrogen, carbon and energy substances.
However, the inability of M9 medium to support swarming is in contrast to
the growth studies in Luria–Bertani medium in which swarmed Proteus
cells expressing fimbrae and pilli were cultured (Latta et al., 1999).
Thus, M9 minimal media is ideal for investigating the physiological roles
of substances in swarming motility of Proteus in vitro. The
observed enhancement of swarming by glutamine, serine and threonine
following supplementation is indicative of a strong contribution of these
amino acids to swarm cell differentiation in these strains. Glutamine has
previously been described as swarming enhancing agent in Serrrattia
liquefaciens, Aeromonas species (Kirov et al., 1986),
Vibrionaceae (McCarter and Silverman, 1990) and Proteus spp
from other environments (Allison et al., 1993). The compliance of our
result to this finding has further validated the universality of glutamine
for this function. Still lacking a define mechanism of action,
studies have strongly implicated glutamine as a chemoattractant and a
positive signal for co-ordinate expression of genes involved
in swarming
motility. Most of
these genes exhibit a high degree of homology in these organisms (Allison
et al., 1991). The capsular polysaccharides of Proteus mirabilis, a
requirement for swarming motility in this organism (Beynon et al., 1992)
have been analyzed to contain branching amino sugars: glucosamine and
galactosamine, which are formed via amino group transfer process.
Glutamine might serve
as a universal amino group donor in this reaction. The involvement of
glutamate could be ruled out since the amino acid was not observed to
enhance swarming in this study. Amino acids such as glycine, asparagines,
aspartate, lysine, histidine also produced relatively lower protease
activity, protein and RNA level in direct relationship with outer colony
zone diameter suggesting their inability to cause marked swarming motility
in these strains. However, the ability of methionine to
support swarming in our strains is a further reflection of the
indispensable requirement of this amino acid in swarming differentiation
in these organisms. Eberl et al. (1999) pointed out that an amino acid
found indispensable for swarming is highly essential in biosynthesis and
operation of the hundreds of flagella produced during swarming
differentiation. In
addition to flagella master control system, cell–to–cell signaling has
also been extensively demonstrated to regulate biosynthetic pathways
involved in signal molecules production and swarm cell differentiation in
organisms that display swarming motility (Eberl et al., 1996a; Glessner et
al., 1998). These signal molecules include serrewatins and acylhomoserine
lactones (AHLs). Methionine could be a precursor of the latter and this is
subject to further investigation. In a few studies, branched chain amino acids
including isoleucine and valine were found to promote swarming motility in
Escherichia coli strain growing on minimal media (Tao et al., 1999). This strain–dependent requirement for swarming–enhancing amino
acids may explain the result obtained for serine in this study. That other amino acids could not support
swarming motility in the strains investigated might be due to
concentration tested and the genetic disposition of these organisms. All
the strains displayed growth in all the supplemented M9 minimal media,
indicating a growth promoting function of these amino acids. Significant activities of extracellular
protease and high concentrations of protein and cellular RNA were also
exhibited by the glutamine, serine and methionine induced swarmed cells.
This observation is in support of the findings of Liaw et al. (2000) in
which p-nitrophenylglycerol an anti-swarming agent was found to inhibit
swarming and expression of virulence factors including Protease in Proteus
mirabilis. Eberl et al. (1996b) also demonstrated high levels of gene
expression in Serratia liquefaciens during hyperflagellation and
swarm cell differentiation. While Allison et al. (1992b) showed that the
uropathogenicity of pathogenic Proteus is coupled to swarming
behavior. The observed high level of protein could be a reflection of
virulence and swarming agents present in the culture media. Those that
have been implicated and which are proteins include urease, haemolysins,
protease, flagellin, fimbrae and pilli.
The
variation in the expression of proteins and cellular RNA level in
glutamine, methionine and serine swarmed cells is an indication that these
amino acids have different modes of swarming promoting action. Finally,
based on the swarming enhancing potentials of serine, glutamine and
methionine, we suggest the exclusion of these amino acids when
supplementing media for the diagnosis of polymicrobial infections
involving Proteus strains from Lagos, Nigeria. However, further
studies are required to elucidate the molecular basis of amino acid – Proteus
interaction that leads to support or negation of swarming motility in this
organism.
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