Bacterial cell wall is an important topic in various fields related to medical sciences. Hopefully, this ppt will provide the necessary information about bacterial cell wall as a whole.

1. BACTERIAL CELL WALL Dr Adya Anwesha, JR,
PGIMER, Chandigarh

2. OUTLINE
A brief history
Introduction
Gram-Positive Cell Walls
Gram-Negative Cell Walls
Atypical organisms
Applied aspects
Conclusion
Mycobacteria
Mycoplasma

3. A BRIEF HISTORY
1884, Hans Christian Gram - “Gram staining” : Gram-positive and Gram-
negative bacteria.
Early 1950s -the chemical composition – speculated chitin or cellulose
(Robert Hooke, 1665)
Salton – wall polymer (now, peptidoglycan)
Park – uridine derivatives (Park’s nucleotide)
Jacob, Hirota, and Spratt - PBPs
In 1951, Corynebacterium diphtheriae - glucosamine and diaminopimelic acid

4. DEFINITION
Rigid and protective outer layer surrounding the cell membrane of bacteria.
Equally important in human and bacteria.
Bacterium contains a well-developed cell structure which is responsible for
some of its unique biological structures and pathogenicity.

5. The cell wall:
structural rigidity
maintains osmotic integrity
cell division
attachment of surface appendages pili, flagella, fimbriae
exposes receptor sites antibiotics or viruses
provides structures for immunological distinction and variation
halt for ligands and proteins for adherence to host cells – virulence determinants
FUNCTIONS

6. CHEMISTRY AND STRUCTURE
10–25 nm thick, 20–25% of dry weight of the cell.
Majority - peptidoglycan, aka murein, backbone of the cell.
Peptidoglycan is a polymer of N-acetyl glucosamine (NAG) and N-acetyl muramic acid
(NAM), linked together by β-1,4 or β-1,6 alternating units, 12 carbohydrates long.
Disaccharide chains - linked together by polypeptide chains (3-8 AA long), attached by
a peptide bond (C=O-NH) to muramic acid carboxyl terminal.

7. Contain unusual amino acids:
• D-alanine and D-glutamic acid - gram-positive bacteria
• Meso-diaminopimelic acid (meso-DAP) or D-lysine - gram-negative bacteria
Tetrapeptides - linked to one another by short peptides forming cross-bridges
D-amino acids in the cell wall - protection from external proteolytic enzymes
Hallmark of PG - glycans are conserved across bacterial species, peptide
stem is often modified and diverse.

8. Cross-links are like a strong web - withstand any kind
of stress.
During synthesis, disaccharide–peptide unit is
inserted into the existing cell wall in the space between
the cytoplasmic membrane and the cell wall.
Enzymes attacking cell wall - lysins.
Lysins attack PG backbone, others attack the peptide
portion or the point where the peptide chain joins the
glycan strands.
Eg., Lysozyme is a lysin that cleaves at the β-1,4
linkage of N-acetylglucosamine.

9. BIOCHEMICAL REACTIONS FOR
PEPTIDOGLYCAN SYNTHESIS
Cell wall biosynthesis is a continuous process.
A three-step mechanism, localized at three locations within a bacterium.
1st stage – cytoplasm, synthesis of the nucleotide sugar-linked precursors
UDP-NAM and UDP-NAG.
2nd stage - cytoplasmic membrane, precursor lipid intermediates are
synthesized: lipid-I and lipid-II.

10. GLUCOSE N-Acetyl glucosamine
UTP
UDP (UDP
NAG)
Phosphoenolpyruvate Enol pyruvate
transferase
(UDP NAM)
L-Ala
D-Glu
L-Lys
UDP-NAM-Tripeptide
UDP-NAM-pentapeptide
L-Ala
D-Ala
D-Ala-D-Ala
Racemase
INSIDE
CYTOPLASM

11. Bactoprenol
UDP-NAM-
pentapeptide
UDP
UDP-NAG
NEAR
CYTOPLASMIC
MEMBRANE

12. Translocation of the lipid-linked precursor from the cytoplasmic side to outer
side of the membrane – peptidoglycan synthase/translocase/flippase.
Studied using - FtsW and RodA proteins – transglycosylation activity.
MurJ - the elusive flippase (Ruiz, 2008; Sham et al., 2014) using substituted cysteine accessibility
method (SCAM).

13. Bactoprenol
Peptidoglycan
synthase/transl
-ocase/flippase
Bactoprenol

14. Final stage - outer side of the cytoplasmic membrane, polymerization of
the new disaccharide-peptide units added to growing PG.
Two steps: transglycosylation and transpeptidation reaction.
Transglycosylation: Reducing end of NAM transferred to C-4 carbon of the
NAG, release of UDP.
UDP - dephosphorylated to yield the lipid carrier bactoprenol.

15. Bactoprenol
Peptidoglycan
synthase
Bactoprenol
(Phosphatase)
P
Dephosphorylation
Recycles Bactoprenol
OUTSIDE
CYTOPLASMIC
MEMBRANE
UDP

16. Transpeptidation: Catalyzed
by a transpeptidase.
Forms an amide bond
between terminal-free amine
group of a stem peptide and a
penultimate D-alanine of a
pentapeptide
Displaces the terminal D-
alanine.
Cleavage reaction provides the
energy, occurs even in the
absence of ATP.
NAG NAM
https://www.sciencedirect.com/science/article/pii/S0223523420302294

17. PEPTIDOGLYCAN TURNOVER
First seen in Bacillus subtilis.
Later, pulse-chased experiments with radioactively labeled cell wall precursors -
all GP as well as GN bacteria carry out a cell wall turnover.
Model for turnover in GPB - inside-to-outside growth, newly synthesized
peptidoglycan is delivered to the cytoplasm in a relaxed form.
Continuous polymerization and cross-linking: high turgor pressure – eventually lysed
by autolysins
How much PG is re-synthesised?

18. Turnover ~ 50% of total cell mass within one
generation.
Hydrolyzed constituents of the cell wall might
be recycled by bacteria (GNB such as E coli)
Cell wall recycling is turned on when GPB
reach the transition to the stationary phase of
growth, not in the exponential growth phase.
In Gram-positive bacteria cell wall recycling is a
crucial step for survival in the stationary phase.

19. GRAM POSITIVE CELL WALL :
COMPOSITION AND STRUCTURE
80 nm thick (range 30-100).
40 - 80% of the dry weight composed of peptidoglycan.
Variety of proteins, polysaccharides and unique molecules
- teichoic acids.
TAs enclose two abundant bacterial cell wall polymers:
(i) LTAs (lipoteichoic acids), which are anchored via lipid
domains in the cytoplasmic membrane, and
(ii) wall TAs (WTAs), which are covalently bound in the
peptidoglycan layers.
https://www.studyandscore.com/studymaterial-detail/cell-wall-of-bacteria-structure-functions-gram-
positive-and-gram-negative-cell-walls

20. Ribitol Teichoic Acids
(Wall TAs)
Glycerol Teichoic
Acids (LTAs)
Associated with Bacterial cell wall
Inner aspect of cell
membrane
Linkage
Covalently linked to
peptidoglycan via the C6
hydroxyl group of NAM
Linked to glycolipids of the
cytoplasm
Location Extends into the cell wall
Linked to the outer lipid
layer of the cell membrane
and extends into the cell
wall
Monomeric Units Ribitol (5-carbon) Glycerol (3-carbon)
Phosphodiester Linkages Present Present

21. KONEMAN Ed7 Page no 190

22. TAs - modified by addition of “R” groups:
Ester-linked D-alanine or D-lysine residues (
O-glycoside-linked glucose, galactose or NAG (Bacillus subtilis)
Teichoic acids:
extra support, stability.
chelators for small ions necessary for cell function and cell wall integrity
cellular interaction and adherence to mucosa and surfaces
role in peptidoglycan synthesis and septum formation during growth and reproduction

23. undergo transformation
antigenic and form the basis for antigenic grouping (group D antigen in
Group D Streptococcus and Enterococci)
LIPOTEICHOIC ACIDS: protect against harmful molecules - antimicrobial peptides
and cationic antibiotics
WALL TEICHOIC ACIDS: for receptor binding and binding to surfaces in
pathogenicity
BOTH: controlling mechanisms for enzyme activities, autolysins and cation
concentrations in PG
mediate biofilm formation and binding to medical devices

24. ADDITIONAL
STRUCTURES IN
BACTERIA
Gram positive + negative

25. Mostly GPB.
Extra layer, single type of protein or
glycoprotein
S-layer proteins are poorly conserved,
have marked differences
Thickness = 5 and 25 nm and
possess identical pores = 2 to 8 nm in
diameter
S-LAYER

26. Functions:
in Archaea - only cell wall component, mechanical stabilization
protection against bacteriophages, low pH, lytic enzymes
adhesin (glycosylated S-layers), attachment sites for periplasmic proteins
inhibit phagocytosis and/or prevent the binding of immunoglobulin and
complement.

27. CAPSULE
Outermost layer, gelatinous polymer
composed of polysaccharides or
polypeptides, or both, surrounds the
entire bacterium with a thick layer.
Bacillus anthracis = polymeric D-glutamic acid
Group A streptococci = hyaluronic acid = D-
glucuronic acid + NAG
It’s called a capsule if the polymer is
firmly attached to the cell wall. If not,
it is called the slime layer.

28. Major functions of capsules in
pathogenic bacteria are
protection against phagocytosis
prevention of complement-mediated bacterial
lysis
contribution to virulence determinants
serotype determinants in Streptococci
Pneumococci: the different capsule
polysaccharides are used as vaccine antigens
enclose bacteria into a biofilm
hydrophilic, prevent dehydration
India Ink, EM
https://journals.asm.org/journal/spectrum

29. Gram-negative bacteria - capsule lies outside the outer membrane
Composition - highly hydrated polyanionic polysaccharides.
determine access of certain molecules to cell membrane
mediate adherence to surfaces
tolerance of desiccation.

30. GRAM NEGATIVE CELL WALL
Thinner than gram positive cell wall, structurally
more complex.
Immediately outside of the cytoplasmic membrane
- periplasmic space, containing degradative
enzymes and specific binding and transport proteins
for vitamins, amino acids, and ions.
A single-unit-thick peptidoglycan layer forms
the outer border of the periplasmic space.
PG - only one layer thick, cross-linking occurs only
to adjacent peptidoglycan strands (not peptides)

31. LPS: high-molecular-weight, complex
glycolipids – unique in GNB
Major surface antigenic determinants (called
somatic or O-antigens)
Responsible for endotoxin activity
Three components:
complex, hydrophobic, lipid portion - lipid A,
core polysaccharide region linking lipid A to the
more external structures,
O-specific (somatic antigen) polysaccharide
side chains, regions of variable biochemical
structure, impart unique serologic identity to gram-
negative species.
Koneman, 7th ed

32. LIPID A
Lipid A - a glucosamine disaccharide,
hydroxyl groups are esterified to β-hydroxy FA
like β-hydroxymyristic acid (C14),
myristomyristic acid, and lauromyristic acid.
Principal component responsible for the
manifestations of endotoxin activity in
patients with gram-negative bacterial sepsis
(fever, shock, vascular collapse, and
haemorrhage).
https://www.lipidmaps.org/resources/lipidweb/lipidw
eb_html/lipids/simple/lipidA/index.htm

33. Two carbohydrates:
3-deoxy-D-mannooctulosonate (formerly called 2-
keto-3-deoxyoctonoic acid [KDO])
heptose
Core KDO - covalent connections between
lipid A and heptose of core polysaccharide.
Additional sugars (NAG, glucose, and
galactose) may be found.
CORE POLYSACCHARIDE
KDO
Heptose
https://www.frontiersin.org/files/Articles/525437

34. Major constituents of GN cell wall
Three major groups:
Porin proteins
Transmembrane proteins
Peripheral proteins
Porin proteins: channels for amino acids,
sugars, ions; doughnut-shaped. Limit passage
of antimicrobial agents.
OUTER MEMBRANE PROTEINS
https://www.researchgate.net/publication/343440297_Periplasmic
_Targets_for_the_Development_of_Effective_Antimicrobials_agains
t_Gram-Negative_Bacteria

35. Transmembrane protein: exoenzyme production and secretion,
protein transport, attachment, binding of antimicrobial agents to their
cell-surface targets (PBPs).
Peripheral proteins: transport of molecules that are too large for porin
entry
Lipoproteins - smallest of OMPs, stabilize the cell wall via covalent
linkage with the peptidoglycan.

36. In some GNB, Neisseria spp., LOS.
Outer leaflet of outer membrane, lower
MW.
Lacks O-antigen polymer
Antigenic variation
LOS -adherence and invasion of host cell,
toxigenicity, pyrogenicity, B-cell mitogenicity
and polyclonal B-cell activation.
https://www.nature.com/articles/nrmicro.2017.169

37. ACID-FAST BACTERIAL CELL
WALL
Mycobacteria - Gram-positive bacteria, high density of lipids in cell wall
prevents accurate Gram staining.
Nocardia and Corynebacterium - also acid-fast.
Three major macromolecules — peptidoglycan, arabinogalactan, and
mycolic acids — building blocks.
Lipids ~ 60% of dry weight.
How is it different from Gram positive cell wall?

38. Cell membrane of mycobacteria -
phosphatidylinositol mannosides and
lipoarabinomannan (LAM).
Single layer PG.
Tetrapeptide bridges – L-ala, D-glu,
meso-DAP.
Some NAM - linked by phosphodiester
bonds to - overlying layer of branched-
chain polysaccharide macromolecules
called arabinogalactans

39. Mycolic acids - large, α-substituted, β-hydroxy fatty acids that occur as esters attached
to cell wall polysaccharides.
Vary in no. of C atoms;
30 carbons (C30) = corynebacteria (corynemycolenic acids)
C50 = Nocardia species (nocardic acids)
C90 or more = genus Mycobacterium.
In Mycobacterium tuberculosis, unique mycolic acid 6,6 ′- dimycolyltrehalose - cord
factor.
MYCOLIC ACIDS

40. Hydrocarbon chains of mycolic acids -
intercalated with other wall associated lipids
and glycolipids (trehalose sulfolipids)
Typified by the principal sulfolipid of M.
tuberculosis 2,3,6,6 ′- tetraacyltrehalose-2
′-sulphate, virulence.
Phosphatidylinositol mannosides and LAM
– noncovalent link b/w cell wall and
membrane
Proteins - biosynthesis and construction of
the cell wall polymers and porins https://www.researchgate.net/figure/Schematic-representation-of-Mycobacterium-showing-the-
main-components-of-the-outer-and_fig1_262533121

41. CAN BACTERIA SURVIVE WITHOUT A
CELL WALL? – CLASS MOLLICUTES
1942 – Eaton’s agent.
Mycoplasma and Ureaplasma – lack cell wall, no PG
Trilaminar unit membrane, small genetic material ~50 kb
Complex lipids for growth
Unaffected by Penicillin
Saprophytic , quickly killed in very high or very low salt concentrations.
Unusually tough membranes - more resistant to rupture.

42. https://www.ncbi.nlm.nih.gov/books/NBK7637/
Electron micrograph of thin-sectioned mycoplasma cells

43. METHODS TO STUDY CELL
WALL
Microscopy techniques:
Electron microscopy (EM)
Atomic force microscopy (AFM)
Cryo-transmission EM (cryo-TEM)
Fluorescent microscope
High Performance Liquid Chromatography (HPLC)
FDAA (Fluorescent-labelled D-amino acids)
X-ray crystallography and liquid state NMR
 Spectroscopic methods:
 CP/MAS - Solid-state Cross-
Polarization Magic Angle Spinning
Carbon-13 Nuclear Magnetic
Resonance
 REDOR – Rotational ECHO
double resonance.
 Radiolabelling.

44. Microscopy techniques – Electron microscopy (EM), but PG needs staining with heavy atoms
for successful imaging.
AFM and cryo-TEM – purified sacculi.
Spatiotemporal dynamics of PG synthesis and remodeling – fluorescent microscopes with
fluorescently labelled antibiotics or lectins; drawbacks – low membrane permeability & toxicity.
Now – FDAA (Fluorescent-labelled D-amino acids) with side chains replaced by
fluorophore.
High Performance Liquid Chromatography (HPLC) – studies in S.aureus show that PG is
highly conserved across a particular species
MICROSCOPY, HPLC

45. AFM images of Staphylococcus aureus

46. https://en.wikipedia.org/wiki/Cryogenic_electron_microscopy
CRYO-TEM in University of Leeds, working principle

47. RADIOLABELLING
Biosynthesis of PG
In-vivo assessment
Submicromolar (fM/pM) detection of CW products and intermediates
Rosenberg, Ohliger, and Wilson developed clinically relevant 11C radiotracers,
metabolically incorporating into both clinically relevant pathogenic GPB and GNB
14C and 11C radionuclides, short-lived (works similar to PET, FDG-18)

48. https://www.cell.com/cell-chemical-biology

49. X-ray crystallography and liquid state NMR, Maya-Martinez et al. structure-function
relationships of PBP4 of Staphylococcus aureus.
Stable isotopes 13C and 15N as probes using spectroscopic methods - uniform
enrichment enhances signal output of the NMR spectrum. Romaniuk and Cegelski et al for
Staphylococcus aureus.
Graphical peaks can reveal ratio of PG to TA.

50. https://en.wikipedia.org/wiki/X-ray_crystallography

51. CP-MAS & REDOR
Rapid assessment of whole-cell composition
Perturbations caused by antiobiotics
13C and 15N – effective probes, (Kim et al., 2014, 2015; Yang et al., 2017)
13C and 15N CP/MAS-NMR - characterize PG and WTA in S. aureus – identification +
quantification by carbon peaks. Romaniuk and Cegelski (2018)
Vancomycin primarily targets transglycosylation over transpeptidation. Cegelski et al. (2002)
REDOR – Amphomycin - induces PG thinning, accumulation of Park’s nucleotide and
decreased alanylation of WTA Singh et al.(2016)

52. APPLIED ASPECTS

53. STAINING PROPERTIES
Simplest way of classifying bacteria.
Gram staining is solely dependent upon the cell wall of bacteria.
Gram positive bacteria retain primary stain (Crystal violet) due to thick PG layer.
CV-Iodine complex - permeability barrier preventing loss of crystal violet.
Gram negative bacteria - thin PG layer and LPS, gets disrupted by decolouriser (Acetone), form
large pores, allowing CV-iodine complex to escape.

54. LIMULUS LYSATE ASSAY
Limulus amoebocyte lysate (LAL) test - detection
of viable and non-viable GNB.
Cell-wall LPS (endotoxins) - gelation of blood
cell (amoebocytes) lysates of the Limulus
polyphemus crab.
Semi-quantitative, chromogenic also (para-
nitroaniline)
Diagnosis of endotoxic shock, fresh meat, milk,
eggs. 1960s : Dr Bang and Dr Levin.

55. M PROTEINS IN
STREPTOCOCCUS
 Cell surface antigen, acid and heat-stable
 2 polypeptide chains with α –helical coiling.
 Anchored to cell mem. through PG.
 N-terminal sequence – Lancefield serologic
c/f for β-hemolytic Strep
 M-proteins – phagocytosis and killing
 Ag in Lancefield grouping:
 Linked to PG: Grp A, B, C, F, G
 To WTA: Grp D KONEMAN, 7th edition

56. ROLE OF GRAM POSITIVE PG
IN VIRULENCE
Lysozyme – muramidase, 1st line of defense
Saliva, tears, urine, mucosal surfaces, airway, blood,
liver and phagocytes
Hydrolyses cell wall, pore-forming.
Bacteria undergoes O-acetylation of N-
acetylmuramoyl residues of PG – inactivation,
increased pathogenesis.
Staphylococcus aureus and Neisseria gonorrhoeae
Currently being investigated as a novel target for anti-
virulence therapies.
https://www.mdpi.com/2079-6382/8/3/94

57. ROLE OF THE GRAM-
NEGATIVE PG IN VIRULENCE
https://journals.asm.org/journal/mmbr

58. 1. Proteins targeting static PG • PG assoc. protein (pal): B.
cenocepacia
• Braun’s lipoprotein
Reduction of virulence - Galleria
mellonella (wax moth)
to 90-fold; impaired host cell
attachment and reduced stimulation
of pro-inflammatory cytokine
secretion
2. Disruption of PBPs • PBP3 (ftsL), cellular division
complex of E. coli
• PBP1a (ponA) and PBP2
(pbpA) in P. aeruginosa
• Chain elongation without
separation, motility
• swarming, biofilm formation
3. Cell division/septations • AmiA, AmiB, AmiC in E. coli Abnormal septa forms, chain
elongation without separation
4. Opening gaps in PG • EtgA in Enterohemorrhagic E. coli T3SS – RBC lysis
5. Host immune response
against PG
• AmpG in S. flexneri permease specific for PG -
NOD1 activation - NFкB

59. ANTIBIOTICS: CELL WALL
SYNTHESIS INHIBITORS
β-Lactam group - Penicillins, Cephalosporins, Monobactams and Carbapenems.
Inhibit “transpeptidase” enzyme, no cross-linking.
These enzymes and related proteins - “Penicillin-binding proteins”
Susceptible bacteria – (+)β-lactam antibiotic - cell wall deficient (CWD) forms
produced. Interior of the bacterium - hyperosmotic, CWD forms swell and burst lysis.
Bactericidal.

60. Peptidoglycan synthesis
inhibitors:
Glycopeptides
Vancomycin
Bacitracin
PG cross-linking inhibitors:
Penicillinase-sensitive penicillins
Penicillinase-resistant penicillins
Carboxypenicillins – Carbenicillin, Ticarcillin
Ureidopenicillins – Piperacillin, Mezlocillin
Cephalosporins
Carbapenems
Monobactams
Penicillin G, V,
Ampicillin,
Amoxicillin
Oxacillin
Cloxacillin
Dicloxacillin
Nafcillin
1st gen: Cefazolin
2nd gen: Cefoxitin
3rd gen: Ceftriaxone
4th gen: Cefepime
5th gen: Ceftaroline,
Ceftabipirole
Imipenem
Ertapenem
Meropenem
Aztreonam

61. ANTIBIOTIC RESISTANCE :
MRSA
Penicillin was the original DOC for S.aureus infections,
resistance was due to acquisition of plasmid-borne
mobile genetic elements encoding for β-lactamase
enzyme.
Methicillin - mecA gene, encoding PBP2a (PBP2’), a
protein with low affinity for β-lactam antibiotics,
conferring resistance to methicillin, nafcillin, oxacillin,
and cephalosporins (chromosomal resistance)
Expression is controlled by two regulatory components :
mecR1-mecI, and the β-lactamase genes blaI, blaRI, and
blaZ, which can downregulate mecA transcription.

62. VRSA
First detected in Japan in 1997 in clinical
isolates
Due to acquisition of the vanA gene
from E. faecium or E. faecalis, which changes
the terminal peptide bond from D-alanyl-
D-alanine to D-alanyl-D-lactate.
https://journals.asm.org/journal/cmr

63. https://journals.asm.org/journal/cmr

64. VISA
VISA - S. aureus isolate with a vancomycin broth MIC of 4 to 8 µg/ml.
S. aureus with reduced vanco susceptibility.
Thicker cell wall, excess D-ala-D-ala.
hVISA: S. aureus with Vanco MIC ≤2µg/ml, but a proportion of the population of
cells are in the vancomycin-intermediate range.

65. VRE
5 phenotypes: vanA, vanB, vanC, vanD, vanE
VanA inducible resistance: by glycopeptides (vancomycin, teicoplanin, avoparcin, and ristocetin)
and by non-glycopeptide agents such as bacitracin, polymyxin B, and robenidine.
vanA gene cluster – transposon Tn1546
vanC : E.casseliflavus, E.gallinarum
Genotypic: vanR, vanS, vanH, vanX, and vanZ induce vanA and B resistance: D-ala-D-lac
vanC: D-ala-D-ser

66. https://journals.asm.org/journal/cmr

67. CONCLUSION
Basic life support of the bacteria.
But not the only one – Capsule and S-
layer
Mediates virulence
Target for antibiotics
Many researches in the field – finished
and ongoing.
https://www.cell.com/cell-chemical-biology