This document discusses various bacterial defense strategies against phages and the human immune system. It describes the CRISPR-Cas system, which allows bacteria to integrate short segments of viral DNA into their genomes to help recognize and destroy that virus in the future. It also discusses restriction modification systems, which use enzymes to cut invading DNA at specific recognition sites to prevent phage replication unless that DNA is properly modified by the host. The document outlines how bacteria avoid and resist phagocytosis through various mechanisms like inhibiting chemotaxis or coating themselves to appear self. It also explains how some bacteria can survive inside phagocytes or escape from phagosomes. Finally, it discusses the ongoing arms race between bacteria and phages as they evolve new defenses and counter-

1. BACTERIAL DEFENSE
STRATEGIES
S.Salomie Jennifer
M.TECH 1ST YEAR
BIO-PHARMACEUTICAL TECHNOLOGY
1

2.  INTRODUCTION
 CRISPR-Cas SYSTEM
 RESTRICTION MODIFICATION
 BACTERIAL DEFENSE AGAINST PHAGOCYTOSIS
 BACTERIA AND PHAGE ARMS RACE
 APPLICATIONS OF DEFENSE SYSTEMS
 CONCLUSION
2

3.  The human immune system's main function is
to protect us against invading bacteria,
viruses, and other pathogens.
 To perform its job, the system has evolved
into a highly complex ensemble of cells,
messengers, and antibody molecules that is
capable of recognizing different pathogens,
defending us against them, and storing
information about them.
3

4.  Even the bacteria themselves are threatened
by pathogens: Certain viruses, the
bacteriophages (literally, "bacteria eaters"),
have become specialized to invade bacterial
cells and proliferate inside of them.
 In order to get rid of these unwanted guests,
many species of bacteria make use of an
arsenal of molecules that works according to
similar principles as an immune system does.
4

5.  Bacteria are constantly subjected to
bacteriophages and other selfish genetic
elements.
 Bacteriophages are viruses that specifically
infect bacteria and the relationship can be
described as a parasitic.
 This is because bacteria are harmed
throughout the phage replication cycle and
often lysed when progeny phage particles
leave the cell .
 Moreover, it is estimated that there are
10^30-10^32 total phage particles on earth,
which outnumber bacteria by 10-fold .
5

6.  This means that phages are found in almost
every environment in which bacteria exist,
making virtually all bacteria susceptible to
phage infection.
 In response to this constant exposure to
phage, bacteria have evolved several diverse
antiviral defense mechanisms.
 These mechanisms include adsorption
blocking, uptake block, abortive infection,
restriction modification and the CRISPR-Cas
system .
6

7. 7

8.  The CRISPR-Cas system is a mechanism that
evolved in bacteria and archea to protect
against genetic element intrusion and
functions similarly to an adaptive immune
system.
 Clustered regularly interspaced short
palindromic repeats (CRISPR), are loci with
several non-continuous direct repeats
separated by stretches of variable sequences
called spacers .
 These repeat and spacer sequences, along
with one or several cas (CRISPR associated)
genes, are key elements present in every
CRISPR-Cas system mechanism
8

9.  The Cas enzyme recognizes DNA molecules
that contain non-self genetic information,
e.g. from bacteriophages, and cleaves them
at specific sites.
 In order to recognize these molecules, a
molecular copy of specific, characteristic
sections of the foreign DNA is required.
 This copy, a kind of "molecular profile" of
bacteriophage DNA and other foreign genetic
material, exists as RNA, an important cellular
building block, which is used, among other
things, as a temporary storage site of genetic
information
9

10.  The template for this profile is stored in the
bacterium's own genes, specifically in those
regions, scientists call CRISPR
 CRISPR which stands for "clustered regularly
interspaced small palindromic repeats" or,
more simply put, the "regular arrangement of
small, symmetric repeats" in the sequence of
the DNA building blocks.
 Together, the enzyme and the profile RNA
constitute the CRISPR-Cas system.
10

11. RohdeStreptococcus pyogenes, shown here while entering a cell,
is one of the germs whose CRISPR-Cas system the scientists have
studied.
11

12.  There are three known types of CRISPR-Cas
systems as well as several diverse subtypes.
 The most commonly used CRISPR-Cas system
is type II, which is naturally found in
Streptococcus thermophiles, a lactic acid
bacterium that is important to the dairy
industry.
 Cas 9 is the key enzyme required for the
CRISPR system to function and has several
enzymatic functions, including endonuclease
and integrase activities.
12

13.  Cas 9 recognizes specific dsDNA sequences in
the phage genome called protospacer
adjacent motive (PAM), and uptakes a
prospacer nucleotide sequence of about 30
bases downstream of the PAM site.
 This sequence is then integrated by Cas9 as a
spacer into the bacterial genome flanked by
two repeat sequences.
 The spacer sequence then gets transcribed
into a precursor CRISPR RNA (pre-crRNA).
13

14.  The pre-crRNA gets processed by an RNase
which is triggered through a transactivating
crRNA (tracrRNA) that is complementary to
the repeat.
 The process happens in the presence of Cas
9, which then associates with the processed
crRNA .
 Upon re-encounter of the same phage
genome, site-specific cleavage by Cas 9
occurs as the target site is determined by
base complementarity between crRNA and
the prospacer in the phage genome
14

15. 15

16. 16

17.  Restriction modification (R-M) is a defense
mechanism which is widely spread among
bacteria .
 There are several types of R-M and all of
these typically involve at least two enzymes,
a restriction endonuclease (REase) and a
methyltransferase (MTase).
 The REase is responsible for the cleavage of
intruding double-stranded DNA, e.g. phage
genomes, through the recognition of the
specific nucleotide restriction sites.
17

18.  Upon genome cleavage the phage is not able
to finish its life cycle.
 Methylation of restriction sites by MTase
protects the host cell genome from cleavage
and REases are categorized as different
restriction endonuclease types depending on
their specific mode of activity
18

19. TYPE CHARACTERISTICS MODE OF ACTIVITY
I MULTI SUB UNIT COMPLEX Cuts DNA at random, away
from recognition site
II GROUPS OF UNRELATED
PROTEINS
Cuts at defined sequence
at/near recognition site
III RESTRICTION AND
MODIFICATION SYSTEMS
Cut outside restriction
sites
Require two restriction
sites in opposite
orientations
IV REQUIRE Mg^2+ FOR
ACTIVITY
Cuts modified DNA
19

20. 20

21. 21

22.  Some pathogenic bacteria are inherently able
to resist the bactericidal components of host
tissues, usually as a function of some
structural property.
 For example, the poly-D-glutamate capsule
of Bacillus anthracis protects the organisms
against action of cationic proteins
(defensins) in sera or in phagocytes.
 The outer membrane of Gram-negative
bacteria is a permeability barrier to lysozyme
and is not easily penetrated by hydrophobic
compounds such as bile salts in the GI tract
that are harmful to the bacteria.
 Pathogenic mycobacteria have a waxy cell
wall that resists attack or digestion by most
tissue bactericides.
22

23.  And intact lipopolysaccharides (LPS) of
Gram-negative pathogens may protect the
cells from complement-mediated lysis or the
action of lysozyme.
 Most successful pathogens, however, possess
additional structural or biochemical features
that allow them to resist the host cellular
defense against them, i.e., the phagocytic
and immune responses.
 If a pathogen breaches the host's surface
defenses, it must then overcome the host's
phagocytic response to succeed in an
infection.
23

24.  Microorganisms invading tissues are first and
foremost exposed to phagocytes.
 Bacteria that readily attract phagocytes and
that are easily ingested and killed are
generally unsuccessful as pathogens.
 In contrast, most bacteria that are
successful as pathogens interfere to some
extent with the activities of phagocytes or in
some way avoid their attention.
24

25.  Bacterial pathogens have devised numerous
and diverse strategies to avoid phagocytic
engulfment and killing.
 Most are aimed at blocking one or more of
the steps in phagocytosis, thereby halting the
process.
 The process of phagocytosis is discussed in
the chapter on Innate Immunity against
bacterial pathogens.
25

26. Bacteria can avoid the attention of phagocytes in a
number of ways.
1. Pathogens may invade or remain confined in
regions inaccessible to phagocytes. Certain
internal tissues (e.g. the lumens of glands, the
urinary bladder) and surface tissues (e.g.
unbroken skin) are not patrolled by phagocytes.
2. Some pathogens are able to avoid provoking an
overwhelming inflammatory response. Without
inflammation the host is unable to focus the
phagocytic defenses.
3. Some bacteria or their products inhibit
phagocyte chemotaxis. For example,
Streptococcal streptolysin (which also kills
phagocytes) suppresses neutrophil chemotaxis,
even in very low concentrations.
26

27. Fractions of Mycobacterium tuberculosis are
known to inhibit leukocyte migration. The
Clostridium toxin also inhibits neutrophil
chemotaxis.
4. Some pathogens can cover the surface of
the bacterial cell with a component which is
seen as "self" by the host phagocytes and
immune system. Such a strategy hides the
antigenic surface of the bacterial cell.
Phagocytes cannot recognize bacteria upon
contact and the possibility of opsonization by
antibodies to enhance phagocytosis is
minimized.
27

28.  For example, pathogenic Staphylococcus
aureus produces cell-bound coagulase and
clumping factor which clots fibrin on the
bacterial surface. Treponema pallidum, the
agent of syphilis, binds fibronectin to its
surface.
 Group A streptococci are able to synthesize a
capsule composed of hyaluronic acid.
Hyaluronic acid is the ground substance
(tissue cement) in connective tissue.
 Some pathogens have or can deposit sialic
acid residues on their surfaces which
prevents opsonization by complement
components and impedes recognition by
phagocytes.
28

29.  Some bacteria survive inside of phagocytes,
either neutrophils or macrophages.
 Bacteria that can resist killing and survive or
multiply inside of phagocytes or other cells
are considered intracellular parasites.
 The intracellular environment of a phagocyte
may be a protective one, protecting the
bacteria during the early stages of infection
or until they develop a full complement of
virulence factors
29

30.  Some intracellular parasites have special
genetically-encoded mechanisms to get
themselves into host cells that are
nonphagocytic.
 Pathogens such as Yersinia, Listeria, E. coli,
Salmonella, Shigella and Legionella possess
complex machinery for cellular invasion and
intracellular survival.
 These systems involve various types of non-
toxin virulence factors.
 Sometimes these factors are referred to as
bacterial invasins.
30

31.  Still other bacteria such as Bordetella
pertussis and Streptococcus pyogenes, have
recently been discovered in the intracellular
habitat of epithelial cells.
 Legionella pneumophila enters mononuclear
phagocytes by depositing complement C3b on
its surfaces and using that host protein to
serve as a ligand for binding to macrophage
cell surfaces.
 After ingestion, the bacteria remain in
vacuoles that do not fuse with lysosomes,
apparently due to the influence of soluble
substances produced by the bacteria.
31

32.  Salmonella bacteria possesses an invasin
operon (inv A - H) that encodes for factors
that regulate their entry into host cells.
 Mutations in the operon yield organisms that
can adhere to target cells without being
internalized.
 This suggests that one or more of the inv
proteins stimulates signal transduction in the
host cell that results engulfment of the
salmonellae.
32

33.  Intracellular parasites survive inside of
phagocytes by virtue of mechanisms which
interfere with the bactericidal activities of the
host cell.
 Some of these bacterial mechanisms include:
1. Inhibition of fusion of the phagocytic
lysosomes (granules) with the phagosome.
 The bacteria survive inside of phagosomes
because they prevent the discharge of lysosomal
contents into the phagosome environment.
 Specifically, phagolysosome formation is
inhibited in the phagocyte.
 This is the strategy employed by Salmonella, M.
tuberculosis, Legionella and the chlamydiae.
33

34. 2. Survival inside the phagolysosome.
 With some intracellular parasites,
phagosome-lysosome fusion occurs, but the
bacteria are resistant to inhibition and killing
by the lysosomal constituents.
 Also, some extracellular pathogens can
resist killing in phagocytes utilizing similar
resistance mechanisms.
 Little is known of how bacteria can resist
phagocytic killing within the phagocytic
vacuole, but it may be due to the surface
components of the bacteria or due to
extracellular substances that they produce
which interfere with the mechanisms of
phagocytic killing.
34

35.  Some examples of how certain bacteria (both
intracellular and extracellular pathogens)
resist phagocytic killing are given below.
 -Mycobacteria (including M. tuberculosis and
Mycobacterium leprae) grow inside phagocytic
vacuoles even after extensive fusion with
lysosomes.
 Mycobacteria have a waxy, hydrophobic cell wall
containing mycolic acids and other lipids, and are
not easily attacked by lysosomal enzymes.
 -Cell wall components (LPS) of Brucella abortus
apparently interfere with the intracellular
bactericidal mechanisms of phagocytes.
35

36. Escape from the phagosome.
 Early escape from the phagosome vacuole is
essential for growth and virulence of some
intracellular pathogens.
 -This is a clever strategy employed by the
Rickettsiae.
 Rickettsia enter host cells in membrane-bound
vacuoles (phagosomes) but are free in the
cytoplasm a short time later, perhaps in as little
as 30 seconds.
 A bacterial enzyme, phospholipase A, may be
responsible for dissolution of the phagosome
membrane.
 -Listeria monocytogenes relies on several
molecules for early lysis of the phagosome to
ensure their release into the cytoplasm.
36

37.  These include a pore-forming hemolysin
(listeriolysin O) and two forms of
phospholipase C.
 Once in the cytoplasm, Listeria induces its
own movement through a remarkable process
of host cell actin polymerization and
formation of microfilaments within a comet-
like tail.
 -Shigella also lyses the phagosomal vacuole
and induces cytoskeletal actin
polymerization for the purpose of
intracellular movement and cell to cell
spread.
37

38. 38

39.  Bacteria and phage are in a constant arm
race of co-evolving defense mechanisms.
 For example, while bacterial defense
mechanisms like CRISPR and restriction
modifications have evolved, phages have
evolved several ways to overcome these.
 In terms of restriction endonucleases, there
are several active and passive ways through
which phage avoid cleavage.
 Passive mechanisms include abundance,
spacing and orientation of restriction sites.
39

40.  Active mechanisms are more specific and in
most cases include specific viral proteins
have evolved to either inhibit restriction site
recognition or proper REase activity
 To overcome the CRISPR-Cas bacterial
defense, phages have evolved both simple
and complex mechanisms.
 In certain cases, a simple point mutation in
the PAM avoids acquisition of spacer
sequence by Cas enzymes.
 Sometimes the whole prospacer and/or PAM
site is deleted from the viral genome, as long
as the deletion doesn’t significantly impair
the phage replication cycle.
40

41.  Other phages harbour complex anti-CRIPSR
proteins encoded in their genome.
 It seems like these proteins inhibit cleavage
of Cas enzymes by preventing proper Cas-
crRNA complex formation.
 Also, recent studies suggest phages have
evolved a CRISPR-Cas system themselves.
 So far, these phage CRISPR-Cas system seem
to form a Cas-crRNA in a similar fashion as
the bacterial one, which can then deactivate
the bacterial CRISPR defense system
41

42. 42

43.  CRISPR and restriction modification are
defense mechanisms to phage infection
which have vast applications in molecular
biology and biotechnology.
 Restriction endonucleases are powerful tools
in molecular biology and several specific
fields, such as metabolic engineering, could
have not been imagined without restriction
enzymes.
 Restriction enzymes of the type II mechanism
are the most common in laboratory
applications and they effectively enable
manipulation of foreign DNA through site
specific cleavage
43

44.  The function of the CRISPR-Cas system is a
fairly new discovery, and there are already
several different applications it is used for.
 Most notably, the CRISPR-Cas system is an
advanced and novel approach in genome
engineering.
 The specificity of the CRISPR system allows
screening for a desired mutations within
genomes and occurs through crRNA:Cas
directed cleavage at targeted sites.
 The CRISPR-Cas system could also be used to
artificially immunize bacterial strains against
specific phages.
44

45.  This has many potential applications in the
food industry, as many processes are
dependent on bacteria, such as the dairy
industry.
 In such industries, engineered phage
immunity could decrease large economic
losses that are caused by phage mediated
infections.
 The CRISPR-Cas has many applications, and
the future of this site specific nuclease will
undoubtedly provide much more
biotechnological advancement in the future.
45

46. 46

47.  Many pathogenic bacteria exist in nature as
multiple antigenic types or serotypes, meaning
that they are variant strains of the same
pathogenic species.
 For example, there are multiple serotypes of
Salmonella enterica based on differences in cell
wall (O) antigens and/or flagellar (H) antigens.
 There are 80 different antigenic types of
Streptococcus pyogenes based on M-proteins on
the cell surface.
 There are over one hundred strains of
Streptococcus pneumoniae depending on their
capsular polysaccharide antigens.
 Based on minor differences in surface structure
chemistry there are multiple serotypes of Vibrio
cholerae, Staphylococcus aureus, Escherichia
coli, Neisseria gonorrhoeae and an assortment of
other bacterial pathogens.
47

48.  Antigenic variation is prevalent among
pathogenic viruses as well.
 If the immunological response is a critical
defense against a pathogen, then being able
to shed old antigens and present new ones to
the immune system might allow infection or
continued invasion by the pathogen to occur.
 Furthermore, the infected host would seem
to be the ideal selective environment for the
emergence of new antigenic variants of
bacteria, providing the organism's other
virulence determinants remain intact.
 Perhaps this explains why many successful
bacterial pathogens exist in a great variety
of antigenic types.
48

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