Functional characterization of VC1929 of Vibrio cholerae El Tor: role in mannose-sensitive haemagglutination, virulence and utilization of sialic acid

Microbiology ◽  
2011 ◽  
Vol 157 (11) ◽  
pp. 3180-3186 ◽  
Author(s):  
Sandeep K. Sharma ◽  
The Su Moe ◽  
Ranjana Srivastava ◽  
Deepak Chandra ◽  
Brahm S. Srivastava
Keyword(s):  
Sialic Acid ◽  
Carbon Source ◽  
Vibrio Cholerae ◽  
Sole Carbon Source ◽  
Minimal Medium ◽  
Adenine Nucleotide ◽  
Functional Characterization ◽  
Acetylneuraminic Acid ◽  
Human Red Blood Cells ◽  
El Tor

The nonadhesive mutant CD11 of Vibrio cholerae El Tor, defective in expression of mannose-sensitive haemagglutinin, lacks a protein when compared with its parent strain. Determination of the amino acid sequence revealed the identity of the protein as the product of VC1929, which is annotated to encode a protein, DctP, involved in the transport of C4-dicarboxylates. We cloned the dctP gene in pUC19 vector and expressed it in mutant CD11. Expression of DctP in the resulting complemented strain restored virulence, adhesive and colonizing capabilities, mannose-sensitive haemagglutination (MSHA) and ability to grow in medium containing sialic acid as a sole carbon source. The mutation in CD11 was caused by insertion of an adenine nucleotide in the reading frame of dctP. Recombinant purified DctP protein showed MSHA of human red blood cells, and protected rabbits against infection by V. cholerae. The protein was localized in membrane and cell wall fractions. The mutant, recombinant CD11 expressing DctP and parent strains were grown in M9 minimal medium in the presence of various carbohydrates (glucose, malate, fumarate, succinate or N-acetylneuraminic acid). The mutant was unable to grow in minimal medium containing N-acetylneuraminic acid (sialic acid) as the sole carbon source whereas the recombinant and parent strains utilized all the sugars tested. It is concluded that DctP is a mannose-sensitive haemagglutinin and a virulence factor and is involved in the utilization of sialic acid.

scholarly journals ESTIMATION OF ANTIBACTERIAL EFFECT ON VIBRIO CHOLERAE (ON THE EXAMPLE OF THE SUBSTANCE ACETYL-N- CYSTEINE–L)

2019 ◽  
pp. 55-57
Author(s):  
O.V. Duvanova ◽  
B.N. Mishan′kin
Keyword(s):  
Minimum Inhibitory Concentration ◽  
Carbon Source ◽  
Vibrio Cholerae ◽  
Sole Carbon Source ◽  
Inhibitory Concentration ◽  
Antibacterial Effect ◽  
Synthetic Medium ◽  
Nutrient Media ◽  
Cholera Vibrio ◽  
El Tor

We studied the effect of the substance acetyl-N-cysteine-L on cholera Vibrio. Its minimum inhibitory concentration for the strains of Vibrio cholerae El Tor O1 and O139 serogroup was 1–2.5 mg/ml in solid nutrient media - Martin's agar (pH 7.6) and LB (pH 7.2), while in Bhascaran synthetic medium, using glucose (0.1per cent) as the sole carbon source, N-acetyl-β-D-glucosamine (0.05 per cent) or colloidal chitin (0.027 per cent) it was reduced to 50–250 μg/ml. The ability of the substance acetyl-N-cysteine-L substance to suppress the activity of the purified enzyme N-acetyl-β-D-glucosaminidase (chitobiasis) was found. Antibacterial effect detected of the substance acetyl-N-cysteine-L against the strains of Vibrio cholerae El Tor O1 and O139 serogroups with different epidemic significance (presence / absence of ctxAB and tcpA genes) indicates the advisability of considering the issue on the possibility of including this substance in composition of solution components used in the rehydration therapy of diarrheal diseases cases.

scholarly journals Sialic Acid Catabolism Confers a Competitive Advantage to Pathogenic Vibrio cholerae in the Mouse Intestine

2009 ◽  
Vol 77 (9) ◽  
pp. 3807-3816 ◽  
Author(s):  
Salvador Almagro-Moreno ◽  
E. Fidelma Boyd
Keyword(s):  
Sialic Acid ◽  
Vibrio Cholerae ◽  
Sialic Acids ◽  
Infant Mouse ◽  
Pathogenic Vibrio ◽  
Important Relationship ◽  
Mouse Intestine ◽  
El Tor ◽  
Mouse Model Of Infection ◽  
Competition Assays

ABSTRACT Sialic acids comprise a family of nine-carbon ketosugars that are ubiquitous on mammalian mucous membranes. However, sialic acids have a limited distribution among Bacteria and are confined mainly to pathogenic and commensal species. Vibrio pathogenicity island 2 (VPI-2), a 57-kb region found exclusively among pathogenic strains of Vibrio cholerae, contains a cluster of genes (nan-nag) putatively involved in the scavenging (nanH), transport (dctPQM), and catabolism (nanA, nanE, nanK, and nagA) of sialic acid. The capacity to utilize sialic acid as a carbon and energy source might confer an advantage to V. cholerae in the mucus-rich environment of the gut, where sialic acid availability is extensive. In this study, we show that V. cholerae can utilize sialic acid as a sole carbon source. We demonstrate that the genes involved in the utilization of sialic acid are located within the nan-nag region of VPI-2 by complementation of E scherichia coli mutants and gene knockouts in V. cholerae N16961. We show that nanH, dctP, nanA, and nanK are highly expressed in V. cholerae grown on sialic acid. By using the infant mouse model of infection, we show that V. cholerae ΔnanA strain SAM1776 is defective in early intestinal colonization stages. In addition, SAM1776 shows a decrease in the competitive index in colonization-competition assays comparing the mutant strain with both O1 El Tor and classical strains. Our data indicate an important relationship between the catabolism of sialic acid and bacterial pathogenesis, stressing the relevance of the utilization of the resources found in the host's environment.

scholarly journals Structural and functional characterization of CMP-N-acetylneuraminate synthetase fromVibrio cholerae

2019 ◽  
Vol 75 (6) ◽  
pp. 564-577
Author(s):  
Sucharita Bose ◽  
Debayan Purkait ◽  
Deepthi Joseph ◽  
Vinod Nayak ◽  
Ramaswamy Subramanian
Keyword(s):  
Sialic Acid ◽  
Vibrio Cholerae ◽  
Active Site ◽  
Drug Targets ◽  
Pathogenic Bacteria ◽  
Pasteurella Multocida ◽  
Bacterial Species ◽  
Functional Characterization ◽  
Gut Colonization

Several pathogenic bacteria utilize sialic acid, including host-derivedN-acetylneuraminic acid (Neu5Ac), in at least two ways: they use it as a nutrient source and as a host-evasion strategy by coating themselves with Neu5Ac. Given the significant role of sialic acid in pathogenesis and host-gut colonization by various pathogenic bacteria, includingNeisseria meningitidis,Haemophilus influenzae,Pasteurella multocidaandVibrio cholerae, several enzymes of the sialic acid catabolic, biosynthetic and incorporation pathways are considered to be potential drug targets. In this work, findings on the structural and functional characterization of CMP-N-acetylneuraminate synthetase (CMAS), a key enzyme in the incorporation pathway, fromVibrio choleraeare reported. CMAS catalyzes the synthesis of CMP-sialic acid by utilizing CTP and sialic acid. Crystal structures of the apo and the CDP-bound forms of the enzyme were determined, which allowed the identification of the metal cofactor Mg2+in the active site interacting with CDP and the invariant Asp215 residue. While open and closed structural forms of the enzyme from eukaryotic and other bacterial species have already been characterized, a partially closed structure ofV. choleraeCMAS (VcCMAS) observed upon CDP binding, representing an intermediate state, is reported here. The kinetic data suggest that VcCMAS is capable of activating the two most common sialic acid derivatives, Neu5Ac and Neu5Gc. Amino-acid sequence and structural comparison of the active site of VcCMAS with those of eukaryotic and other bacterial counterparts reveal a diverse hydrophobic pocket that interacts with the C5 substituents of sialic acid. Analyses of the thermodynamic signatures obtained from the binding of the nucleotide (CTP) and the product (CMP-sialic acid) to VcCMAS provide fundamental information on the energetics of the binding process.

scholarly journals Dynamics of sodium dodecyl sulfate utilization andantibiotic susceptibility of strain Pseudomonas sp. ATCC19151

2009 ◽  
Vol 61 (2) ◽  
pp. 159-164 ◽  
Author(s):  
B. Jovcic ◽  
Jelena Begovic ◽  
Jelena Lozo ◽  
L. Topisirovic ◽  
Milan Kojic
Keyword(s):  
Carbon Source ◽  
Sodium Dodecyl Sulfate ◽  
Sole Carbon Source ◽  
Minimal Medium ◽  
Sodium Dodecyl ◽  
Pseudomonas Sp ◽  
Gene Encoding ◽  
Dodecyl Sulfate ◽  
Minimal Media ◽  
Growth Ability

Pseudomonas sp. ATCC19151 harbors a gene encoding a putative alkylsulfatase (sdsA). Here we report a growth ability of this strain in minimal media containing 0.5, 0.75, and 1% sodium dodecyl sulfate as the sole carbon source. The most prominent growth was detected for the minimal medium with 0.5% SDS, so this concentration of SDS was used to monitor Pseudomonas sp. ATCC19151 SDS biodegradation dynamics. Bacterial growth coincided with the disappearance of SDS. Antibiotic susceptibility was tested as well. Pseudomonas sp. ATCC19151 was resistant to six out of nine tested antibiotics, including ampicillin, tetracycline, chloramphenicol, tobramycin, nalidixic acid, and gentamycin.

scholarly journals Sialic Acid Transport Contributes to Pneumococcal Colonization

2010 ◽  
Vol 79 (3) ◽  
pp. 1262-1269 ◽  
Author(s):  
Carolyn Marion ◽  
Amanda M. Burnaugh ◽  
Shireen A. Woodiga ◽  
Samantha J. King
Keyword(s):  
Sialic Acid ◽  
Carbon Source ◽  
Sole Carbon Source ◽  
Upper Respiratory Tract ◽  
Acid Transport ◽  
Upper Respiratory ◽  
Pneumococcal Colonization ◽  
Free Carbohydrates ◽  
Airway Colonization

ABSTRACTStreptococcus pneumoniaeis a major cause of pneumonia and meningitis. Airway colonization is a necessary precursor to disease, but little is known about how the bacteria establish and maintain colonization. Carbohydrates are required as a carbon source for pneumococcal growth and, therefore, for colonization. Free carbohydrates are not readily available in the naso-oropharynx; however, N- and O-linked glycans are common in the airway. Sialic acid is the most common terminal modification on N- and O-linked glycans and is likely encountered frequently byS. pneumoniaein the airway. Here we demonstrate that sialic acid supports pneumococcal growth when provided as a sole carbon source. Growth on sialic acid requires import into the bacterium. Three genetic regions have been proposed to encode pneumococcal sialic acid transporters: one sodium solute symporter and two ATP binding cassette (ABC) transporters. Data demonstrate that one of these,satABC, is required for transport of sialic acid. AsatABCmutant displayed significantly reduced growth on both sialic acid and the human glycoprotein alpha-1. The importance ofsatABCfor growth on human glycoprotein suggests that sialic acid transport may be importantin vivo. Indeed, thesatABCmutant was significantly reduced in colonization of the murine upper respiratory tract. This work demonstrates thatS. pneumoniaeis able to use sialic acid as a sole carbon source and that utilization of sialic acid is likely important during pneumococcal colonization.

scholarly journals DsdX Is the Second d-Serine Transporter in Uropathogenic Escherichia coli Clinical Isolate CFT073

2006 ◽  
Vol 188 (18) ◽  
pp. 6622-6628 ◽  
Author(s):  
Andrew T. Anfora ◽  
Rodney A. Welch
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Carbon Source ◽  
Clinical Isolate ◽  
Sole Carbon Source ◽  
Minimal Medium ◽  
Amino Acid Sequence Similarity ◽  
Predicted Amino Acid Sequence ◽  
E Coli ◽  
K 12

ABSTRACTd-Serine is an amino acid present in mammalian urine that is inhibitory toEscherichia colistrains lacking a functionaldsdAgene. Counterintuitively, adsdAstrain ofE. coliclinical isolate CFT073 hypercolonizes the bladder and kidneys of mice relative to wild type during a coinfection in the murine model of urinary tract infection. We are interested in the mechanisms for uptake ofd-serine in CFT073.d-Serine entersE. coliK-12 via CycA, thed-alanine transporter andd-cycloserine sensitivity locus. CFT073cycAcan grow on minimal medium withd-serine as a sole carbon source. ThedsdXgene of thedsdCXAlocus is a likely candidate for an additionald-serine transporter based on its predicted amino acid sequence similarity to gluconate transporters. In minimal medium, CFT073dsdXcan grow ond-serine as a sole carbon source; however, CFT073dsdX cycAcannot. Additionally, CFT073dsdXA cycAis not sensitive to inhibitory concentrations ofd-serine during growth on glycerol andd-serine minimal medium.d-[14C]serine uptake experiments with CFT073dsdX cycAharboringdsdXorcycArecombinant plasmids confirm thatd-serine is able to enterE. colicells via CycA or DsdX. In whole-celld-[14C]serine uptake experiments, DsdX has an apparentKmof 58.75 μM and aVmaxof 75.96 nmol/min/mg, and CycA has an apparentKmof 82.40 μM and aVmaxof 58.90 nmol/min/mg. Onlyd-threonine marginally inhibits DsdX-mediatedd-serine transport, whereasd-alanine, glycine, andd-cycloserine inhibit CycA-mediatedd-serine transport. DsdX or CycA is sufficient to transport physiological quantities ofd-serine, but DsdX is ad-serine-specific permease.

scholarly journals Transport and Catabolism of Carbohydrates by Neisseria meningitidis

2016 ◽  
Vol 26 (5) ◽  
pp. 320-332 ◽  
Author(s):  
Meriem Derkaoui ◽  
Ana Antunes ◽  
Jamila Nait Abdallah ◽  
Sandrine Poncet ◽  
Alain Mazé ◽  
...  
Keyword(s):  
Carbon Source ◽  
Transgenic Mice ◽  
Neisseria Meningitidis ◽  
Pentose Phosphate Pathway ◽  
Sole Carbon Source ◽  
Minimal Medium ◽  
Pentose Phosphate ◽  
Key Enzymes ◽  
The Real ◽  
Genes Encoding

We identified the genes encoding the proteins for the transport of glucose and maltose in <i>Neisseria meningitidis</i> strain 2C4-3. A mutant deleted for <i>NMV_1892</i><i>(glcP)</i> no longer grew on glucose and deletion of <i>NMV_0424</i><i>(malY)</i> prevented the utilization of maltose. We also purified and characterized glucokinase and α-phosphoglucomutase, which catalyze early catabolic steps of the two carbohydrates. <i>N. meningitidis</i> catabolizes the two carbohydrates either via the Entner-Doudoroff (ED) pathway or the pentose phosphate pathway, thereby forming glyceraldehyde-3-P and either pyruvate or fructose-6-P, respectively. We purified and characterized several key enzymes of the two pathways. The genes required for the transformation of glucose into gluconate-6-P and its further catabolism via the ED pathway are organized in two adjacent operons. <i>N. meningitidis</i> also contains genes encoding proteins which exhibit similarity to the gluconate transporter <i>(NMV_2230)</i> and gluconate kinase <i>(NMV_2231)</i> of Enterobacteriaceae and Firmicutes. However, gluconate might not be the real substrate of <i>NMV_2230</i> because <i>N. meningitidi</i>s was not able to grow on gluconate as the sole carbon source. Surprisingly, deletion of <i>NMV_2230</i> stimulated growth in minimal medium in the presence and absence of glucose and drastically slowed the clearance of <i>N. meningitidis</i> cells from transgenic mice after intraperitoneal challenge.

scholarly journals Sialic Acid (N-Acetyl Neuraminic Acid) Utilization by Bacteroides fragilis Requires a Novel N-Acetyl Mannosamine Epimerase

2009 ◽  
Vol 191 (11) ◽  
pp. 3629-3638 ◽  
Author(s):  
Christopher Brigham ◽  
Ruth Caughlan ◽  
Rene Gallegos ◽  
Mary Beth Dallas ◽  
Veronica G. Godoy ◽  
...  
Keyword(s):  
Energy Source ◽  
Sialic Acid ◽  
Carbon Source ◽  
Mutant Strain ◽  
Sole Carbon Source ◽  
Binding Proteins ◽  
Bacteroides Fragilis ◽  
Neuraminic Acid ◽  
The Third

ABSTRACT We characterized the nanLET operon in Bacteroides fragilis, whose products are required for the utilization of the sialic acid N-acetyl neuraminic acid (NANA) as a carbon and energy source. The first gene of the operon is nanL, which codes for an aldolase that cleaves NANA into N-acetyl mannosamine (manNAc) and pyruvate. The next gene, nanE, codes for a manNAc/N-acetylglucosamine (NAG) epimerase, which, intriguingly, possesses more similarity to eukaryotic renin binding proteins than to other bacterial NanE epimerase proteins. Unphosphorylated manNAc is the substrate of NanE, while ATP is a cofactor in the epimerase reaction. The third gene of the operon is nanT, which shows similarity to the major transporter facilitator superfamily and is most likely to be a NANA transporter. Deletion of any of these genes eliminates the ability of B. fragilis to grow on NANA. Although B. fragilis does not normally grow with manNAc as the sole carbon source, we isolated a B. fragilis mutant strain that can grow on this substrate, likely due to a mutation in a NAG transporter; both manNAc transport and NAG transport are affected in this strain. Deletion of the nanE epimerase gene or the rokA hexokinase gene, whose product phosphorylates NAG, in the manNAc-enabled strain abolishes growth on manNAc. Thus, B. fragilis possesses a new pathway of NANA utilization, which we show is also found in other Bacteroides species.

scholarly journals Structural and Functional Characterization of IS1358 from Vibrio cholerae

1998 ◽  
Vol 180 (23) ◽  
pp. 6101-6106 ◽  
Author(s):  
Sandrine Dumontier ◽  
Patrick Trieu-Cuot ◽  
Patrick Berche
Keyword(s):  
Vibrio Cholerae ◽  
Insertion Sequence ◽  
Functional Characterization ◽  
Insertion Site ◽  
Kanamycin Resistance ◽  
Conjugative Plasmid ◽  
Multiple Copies ◽  
Dna Fragment ◽  
El Tor

ABSTRACT The new epidemic serovar O139 of Vibrio cholerae has emerged from the pandemic serovar O1 biotype El Tor through the replacement of a 22-kbp DNA region by a 40-kbp O139-specific DNA fragment. This O139-specific DNA fragment contains an insertion sequence that was described previously (U. H. Stroeher, K. E. Jedani, B. K. Dredge, R. Morona, M. H. Brown, L. E. Karageorgos, J. M. Albert, and P. A. Manning, Proc. Natl. Acad. Sci. USA 92:10374–10378, 1995) and designated IS1358 O139. We studied the distribution of the IS1358 element in strains from various serovars by Southern analysis. Its presence was detected in strains from serovars O1, O2, O22, O139, and O155 but not in strains from serovars O15, O39, and O141. Furthermore, IS1358 was present in multiple copies in strains from serovars O2, O22, and O155. We cloned and sequenced four copies of IS1358 from V. cholerae O22 and one copy from V. cholerae O155. A comparison of their nucleotide sequences with those of O1 and O139 showed that they were almost identical. We constructed a transposon consisting of a kanamycin resistance gene flanked by two directly oriented copies of IS1358 to study the functionality of this element. Transposition of this element from a nonmobilizable plasmid onto the conjugative plasmid pOX38-Gen was detected in an Escherichia coli recA donor at a frequency of 1.2 × 10−8. Sequence analysis revealed that IS1358 duplicates 10 bp at its insertion site.

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